Compositions comprising bacterial strains

ABSTRACT

The invention provides a composition comprising a bacterial strain of the genus Megasphaera, for use in stimulating the immune system in subject.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.17/013,026, filed Sep. 4, 2020, which is a continuation of InternationalApplication No. PCT/EP2019/062238, filed May 13, 2019, which claims thebenefit of Great Britain Application No. 1820256.4, filed Dec. 12, 2018,Great Britain Application No. 1820264.8, filed Dec. 12, 2018, GreatBritain Application No. 1817642.0, filed Oct. 29, 2018, Great BritainApplication No. 1813460.1, filed Aug. 17, 2018, Great BritainApplication No. 1810386.1, filed Jun. 25, 2018, European PatentApplication No. 18178136.0, filed Jun. 15, 2018 and European ApplicationNo. 18171893.3, filed May 11, 2018; all of which are hereby incorporatedby reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 5, 2020, isnamed 56708_738_301_SL.txt and is 15,238 bytes in size.

TECHNICAL FIELD

This invention is in the field of compositions comprising bacterialstrains isolated from the mammalian digestive tract and the use of suchcompositions in the treatment of disease, in particular cancer, and inparticular in stimulating the immune system in the treatment of disease.

BACKGROUND TO THE INVENTION

The human intestine is thought to be sterile in utero, but it is exposedto a large variety of maternal and environmental microbes immediatelyafter birth. Thereafter, a dynamic period of microbial colonization andsuccession occurs, which is influenced by factors such as delivery mode,environment, diet and host genotype, all of which impact upon thecomposition of the gut microbiota, particularly during early life.Subsequently, the microbiota stabilizes and becomes adult-like [1]. Thehuman gut microbiota contains more than 500-1000 different phylotypesbelonging essentially to two major bacterial divisions, theBacteroidetes and the Firmicutes [2]. The successful symbioticrelationships arising from bacterial colonization of the human gut haveyielded a wide variety of metabolic, structural, protective and otherbeneficial functions. The enhanced metabolic activities of the colonizedgut ensure that otherwise indigestible dietary components are degradedwith release of by-products providing an important nutrient source forthe host. Similarly, the immunological importance of the gut microbiotais well-recognized and is exemplified in germfree animals which have animpaired immune system that is functionally reconstituted following theintroduction of commensal bacteria [3-5].

Dramatic changes in microbiota composition have been documented ingastrointestinal disorders such as inflammatory bowel disease (IBD). Forexample, the levels of Clostridium cluster XIVa bacteria are reduced inIBD patients whilst numbers of E. coli are increased, suggesting a shiftin the balance of symbionts and pathobionts within the gut [6-9].Interestingly, this microbial dysbiosis is also associated withimbalances in T effector cell populations.

In recognition of the potential positive effect that certain bacterialstrains may have on the animal gut, various strains have been proposedfor use in the treatment of various diseases (see, for example,[10-13]). Also, certain strains, including mostly Lactobacillus andBifidobacterium strains, have been proposed for use in treating variousinflammatory and autoimmune diseases that are not directly linked to theintestines (see [14] and [15] for reviews). However, the relationshipbetween different diseases and different bacterial strains, and theprecise effects of particular bacterial strains on the gut and at asystemic level and on any particular types of diseases, are poorlycharacterised.

WO2015038731 discusses a method of treating colon cancer by disruptionof a colonic biofilm by administration of an antimicrobial agent or aprobiotic agent. The application lists a large number of bacteria thatmay be used in a probiotic but provides no demonstration of the efficacyof any of the bacteria in the treatment of colon cancer. Instead, thisapplication focuses on the diagnostic potential of biofilms incolorectal cancer.

EMBL database accession number XP002787383 provides a 16S rRNA genesequence of a proposed Megasphaera sp, while EMBL database accessionnumber XP002787384 provides a 16S rRNA gene of a Megasphaeramassiliensis strain. These documents detail the genomic analysis ofisolated strains and provide no guidance towards the therapeutic benefitof Megasphaera.

Ahmed et al (submitted to Frontiers Cellular Neuroscience) considers invitro characterisation of gut microbiota-derived bacterial strains.

There is a requirement in the art for new methods of treating diseases.There is also a requirement for the potential effects of gut bacteria tobe characterised so that new therapies using gut bacteria can bedeveloped.

SUMMARY OF THE INVENTION

The inventors have developed new compositions comprising a bacterialstrain of the genus Megasphaera that can be used in stimulating theimmune system and treating and preventing disease, in particular cancer.

The invention therefore provides a composition comprising a bacterialstrain of the genus Megasphaera, for use in stimulating the immunesystem in subject. Preferably, the bacterial strain is of the speciesMegasphaera massiliensis.

In further aspects, the invention provides a composition comprising abacterial strain of the genus Megasphaera, for use in treating orpreventing cancer, such as metastatic melanoma, breast cancer, ovariancancer, cervical cancer, neuroblastoma, glioblastoma, carcinoma, lungcancer, chronic lymphocyte leukemia, prostate cancer, lymphoma and/orgastric cancer. In further aspects, the invention provides a compositioncomprising a bacterial strain of the genus Megasphaera, for use intreating or preventing cancer, such as colorectal cancer and/orhaematological malignancies.

In further aspects, the invention provides a composition comprising abacterial strain of the genus Megasphaera, for use in treating,preventing or delaying immunosenescence.

In further aspects, the invention provides a composition comprising abacterial strain of the genus Megasphaera, for use as a vaccineadjuvant.

In further aspects, the invention provides a composition comprising abacterial strain of the genus Megasphaera, for use in enhancing a celltherapy, such as CAR-T.

Preferably, the bacteria used in the invention is the strain depositedunder accession number 42787 at NCIMB.

Further numbered embodiments of the invention are provided below:

-   -   1. A composition comprising a bacterial strain of the genus        Megasphaera, for use in stimulating the immune system in        subject.    -   2. The composition of embodiment 1, for use in treating or        preventing cancer, such as metastatic melanoma, breast cancer,        ovarian cancer, cervical cancer, neuroblastoma, glioblastoma,        carcinoma, lung cancer, chronic lymphocyte leukemia, prostate        cancer, lymphoma, gastric cancer, colorectal cancer and/or        haematological malignancies.    -   3. The composition for use according to embodiment 2, wherein        the composition has histone deacetylase inhibitory activity.    -   4. The composition for use according to embodiment 2 or        embodiment 3, wherein the composition up-regulates        pro-inflammatory cytokines.    -   5. The composition for use according to any one of embodiments        2-4, for use in reducing gut barrier permeability.    -   6. The composition of embodiment 1, for use in treating,        preventing or delaying immunosenescence.    -   7. The composition of embodiment 1, for use as a vaccine        adjuvant.    -   8. The composition of embodiment 1, for use in enhancing a cell        therapy, such as CAR-T.    -   9. The composition of any preceding embodiment, for use in        increasing the expression level and/or activity of Caspase 3,        MAP2, IL-1β, IL-23 and/or TNF-α.    -   10. The composition of any preceding embodiment, for use in a        method of selectively decreasing the number and/or percentage of        Tregs in a cell population.    -   11. The composition of any preceding embodiment, wherein the        bacterial strain has a 16S rRNA sequence that is at least 95%,        96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16S rRNA        sequence of a bacterial strain of the genus Megasphaera.    -   12. The composition of any preceding embodiment, wherein the        bacterial strain has a 16s rRNA gene sequence that is at least        95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to any one of        SEQ ID NOs:8, 9, 10, 11 or 12 or wherein the bacterial strain        has a 16s rRNA gene sequence represented by any one of SEQ ID        NOs:8, 9, 10, 11 or 12.    -   13. The composition of any preceding embodiment, wherein the        bacterial strain is of Megasphaera massiliensis.    -   14. The composition of any preceding embodiment, wherein the        bacterial strain has a 16s rRNA gene sequence that is at least        95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:1        or wherein the bacterial strain has a 16s rRNA gene sequence        represented by SEQ ID NO:1.    -   15. The composition of any preceding embodiment, wherein the        bacterial strain is the strain deposited under accession number        42787 at NCIMB.    -   16. The composition of any preceding embodiment, wherein the        composition is for oral administration.    -   17. The composition of any preceding embodiment, wherein the        composition comprises one or more pharmaceutically acceptable        excipients or carriers.    -   18. The composition of any preceding embodiment, wherein the        bacterial strain is lyophilised.    -   19. A food product comprising the composition of any preceding        embodiment, for the use of any preceding claim.    -   20. A method of treating or preventing a disease or condition        associated with reduced immunostimulation, comprising        administering a composition comprising a bacterial strain of the        genus Megasphaera to a patient in need thereof.    -   21. A composition comprising a cell of the bacterial strain        defined in any of embodiments 1 to 16, wherein the cell        expresses one or more heterologous antigens.    -   22. The composition according to embodiment 21, wherein the cell        presents the one or more heterologous antigens.    -   23. The composition according to embodiment 21 or embodiment 22,        for use as a vaccine.    -   24. A cell of the bacterial strain defined in any of embodiments        1 to 18, wherein the cell expresses one or more heterologous        antigens.    -   25. The cell according to embodiment 24, wherein the cell        presents the one or more heterologous antigens.    -   26. The cell according to embodiment 24 or embodiment 25, for        use as a vaccine.    -   27. A bacterial strain for use in therapy, wherein the bacterial        strain has a 16S rRNA sequence that is at least 95%, 96%, 97%,        98%, 99%, 99.5% or 99.9% identical to any one of SEQ ID NOs:8,        9, 10, 11 or 12.    -   28. A bacterial strain having the 16S rRNA sequence represented        by any one of SEQ ID NOs: 8, 9, 10, 11 or 12 for use in therapy.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1B: Levels of β3 Tubulin expression: immunostaining and cellimaging (FIG. 1A); immunoblotting (FIG. 1B)

FIGS. 2A-2C: Levels of MAP2 expression: immunostaining and cell imaging(FIG. 2A); immunoblotting (FIG. 2B); fold change in expression (FIG. 2C)

FIG. 3 : Change in DRD2 expression

FIG. 4 : Change in Casp3 expression

FIG. 5 : Change in cell viability FIGS. 6A-6H: Cell phenotyping of CD4 Thelper cells (FIG. 6A); CD4+ activated cells (FIG. 6B); Tregs cells(FIG. 6C); CD8 cytotoxic T cells (FIG. 6D); CD8+ activated cells (FIG.6E); B cells (FIG. 6F); CD8/Treg ratio (FIG. 6G); Activated CD8/Tregratio (FIG. 6H).

FIGS. 7A-7I: Cytokine analysis of IL-1β (FIG. 7A); TNF-α (FIG. 7B);IL-23 (FIG. 7C); IL-6 (FIG. 7D); MIP-3α (FIG. 7E); CXCL9 (FIG. 7F);MCP-1 (FIG. 7G); IL-10 (FIG. 7H); GM-CSF (FIG. 7I).

FIGS. 8A-8C: Gating strategy used to analyse the different population ofimmune cells (CD4, CD8 and CD19+ cells; FIG. 8A, FIG. 8B, FIG. 8C) byFlow Cytometry for the data presented in FIG. 6 .

FIG. 9 : Secretion of Interleukin-8 (IL-8)).

FIG. 10 : Change in histone deacetylase (HDAC) activity

FIG. 11A: Strain-induced changes in whole cell and cell lysate histonedeacetylase activity; FIG. 11B: metabolite production by strains; FIG.11C: acid-induced changes in histone deacetylase activity.

FIG. 12A: HDAC1 inhibition; FIG. 12B: HDAC2 inhibition; FIG. 12C: HDAC3inhibition.

FIG. 13A: Inhibition of Class I HDACs; FIG. 13B: inhibition of HDAC1;FIG. 13C: inhibition of HDAC2; FIG. 13D: inhibition of HDAC3.

FIGS. 14A-14F: Effects on intestinal barrier function. FIG. 14A: IL-8secretion; FIG. 14B: Occludin, Villin, TJP1 and TJP2 mRNA expression;FIG. 14C: TJP1 mRNA expression; FIG. 14D: Occludin mRNA expression; FIG.14E: Permeability in the Ileum; FIG. 14F: Permeability in the Colon.

FIG. 15 : Changes in Hippocampal expression of Toll-like Receptor 4(TLR-4).

FIG. 16 : Changes in Hippocampal expression of TNF-α.

FIG. 17 : Changes in Hippocampal expression of Interleukin-1β (IL-1β).

FIG. 18 : Changes in Hippocampal expression of Interleukin-6 (IL-6).

FIG. 19 : Changes in Hippocampal expression of CD11b.

FIG. 20 : Changes in Amygdala expression of TLR-4.

FIG. 21 : Changes in Amygdala expression of CD11b.

FIG. 22 : Changes in Amygdala expression of IL-6.

FIG. 23 : Changes in Prefrontal Cortex expression of TLR-4.

FIG. 24 : Changes in Prefrontal Cortex expression of CD11b.

FIG. 25 : Changes in Prefrontal Cortex Expression of IL-6.

FIG. 26 : Effect on Interferon-γ production from mouse splenocytes frommice administered MRx0029.

FIG. 27 : Effect on IL-1β production from mouse splenocytes from miceadministered MRx0029.

FIG. 28 : Effect on IL-6 production from mouse splenocytes from miceadministered MRx0029.

FIG. 29 : Effect on TNF-α production from splenocytes from miceadministered MRx0029.

FIG. 30 : Effect on CXCL1 production from splenocytes from miceadministered MRx0029.

FIG. 31 : Gene expression of MAP2 in the SKMEL2 cell line followingvarious treatments, relative to GAPDH. “YCFA”=YCFA+

FIG. 32 : Clonogenic survival of the SKMEL2 cell line following varioustreatments. “YCFA”=YCFA+

FIG. 33 : Soft agar growth of the SKMEL2 cell line following varioustreatments. “YCFA”=YCFA+

FIG. 34 : ERK signalling (phosphorylated ERK1 and 2 (p44 and p42)/totalERK) in the SKMEL2 cell line following various treatments. “YCFA”=YCFA+

FIG. 35 : Gene expression of MAP2 in the SKMEL28 cell line followingvarious treatments, relative to GAPDH. “YCFA”=YCFA+

FIG. 36 : Clonogenic survival of the SKMEL28 cell line following varioustreatments. “YCFA”=YCFA+

FIG. 37 : Soft agar growth of the SKMEL28 cell line following varioustreatments. “YCFA”=YCFA+

FIG. 38 : ERK signalling (phosphorylated ERK1 and 2 (p44 and p42)/totalERK) in the SKMEL28 cell line following various treatments. “YCFA”=YCFA+

FIG. 39 : Gene expression of MAP2 in the SKMEL31 cell line followingvarious treatments, relative to GAPDH. “YCFA”=YCFA+

FIG. 40 : Clonogenic survival of the SKMEL31 cell line following varioustreatments. “YCFA”=YCFA+

FIG. 41 : Soft agar growth of the SKMEL31 cell line following varioustreatments. “YCFA”=YCFA+

FIG. 42 : ERK signalling (phosphorylated ERK1 and 2 (p44 and p42)/totalERK) in the SKMEL31 cell line following various treatments. “YCFA”=YCFA+

FIG. 43 : Gene expression of MAP2 in the 451Lu cell line followingvarious treatments, relative to GAPDH. “YCFA”=YCFA+

FIG. 44 : Clonogenic survival of the 451Lu cell line following varioustreatments. “YCFA”=YCFA+

FIG. 45 : Soft agar growth of the 451Lu cell line following varioustreatments. “YCFA”=YCFA+

FIG. 46 : ERK signalling (phosphorylated ERK1 and 2 (p44 and p42)/totalERK) in the 451Lu cell line following various treatments. “YCFA”=YCFA+

FIG. 47 : Gene expression of MAP2 in the HT-29 cell line followingvarious treatments, relative to GAPDH. “YCFA”=YCFA+

FIG. 48 : Clonogenic survival of the HT-29 cell line following varioustreatments. “YCFA”=YCFA+

FIG. 49A: Soft agar growth of the HT-29 cell line following varioustreatments. “YCFA”=YCFA+

FIG. 49B: Soft agar growth of the HT-29 cell line following varioustreatments (photograph of agar plates). “YCFA”=YCFA+

FIG. 50 : ERK signalling (phosphorylated ERK1 and 2 (p44 and p42)/totalERK) in the HT29 cell line following various treatments. “YCFA”=YCFA+

FIG. 51 : Overview of the MAP-kinase pathway (from [72]).

FIGS. 52A and 52B: GPR109a RNA expression in differentiated Caco-2 cells(FIG. 52A) without, and (FIG. 52B) with phorbolmyristate treatment inaddition to MRx0029. “YCFA”=YCFA+

FIGS. 53A and 53B: Induction of IL-8 secretion from HT29 cells by (FIG.53A) MRx0029 with conditioned media and (FIG. 53B) MRx0029 alone.

FIG. 54 : Metabolite analysis for Megasphaera massiliensis strain NCIMB42787.

FIG. 55 : Valeric acid production in the supernatant for MRx0029 andreference Megasphaera massiliensis strains.

FIG. 56 : organic acid production and consumption by MRx0029 andreference Megasphaera massiliensis strains.

FIG. 57 : Suppression of NSE/Enolase 2 by MRX029. “YCFA”=YCFA+.

FIG. 58 : Organic acid production and consumption by NCIMB 42787, NCIMB43385, NCIMB 43388 and NCIMB 43389.

FIG. 59 : Upregulation of IL-6 secretion in U373 cells by NCIMB 42787and other deposited strains (n=3).

FIG. 60 : Suppression of Enolase 2 by NCIMB 42787, NCIMB 43385, NCIMB43388, NCIMB 43389, NCIMB 43386 and NCIMB 43387.

FIG. 61A: NCIMB 42787 and other deposited increase MAP2 expression;FIGS. 61B and 61C: Modulation of cytokine levels and NFκB-AP1 promoterby NCIMB 42787.

FIGS. 62A-62C: NCIMB 42787 produces butyric, valeric and hexanoic acid.FIG. 62A: SCFA and MCFA; FIG. 62B: Succinic acid, 4-Hydroxy-PhenylAcetic Acid; FIG. 62C: SCFA standards vs NCIMB 42787.

FIG. 63 : Immunostimulatory activity of metabolites produced by NCIMB42787.

FIGS. 64A and 64B: Analysis of role of metabolites in immunostimulatoryactivity of NCIMB 42787. FIG. 64A: IL-8; FIG. 64B: IL-6.

FIG. 65 : Megasphaera strain NCIMB 43387 affects colonic IDO-1 mRNAexpression in BALB/c mice.

FIG. 66 : Megasphaera strains NCIMB 43385 and NCIMB 43387 affect colonicTph1 mRNA expression in BALB/c mice.

FIGS. 67A and 67B: Megasphaera strain NCIMB 43385 modulates IFNγ andIL-6 production upon ConA stimulation of splenocytes from BALB/c mice.FIG. 67A: IFNγ; FIG. 67B: IL-6.

FIG. 68 : Megasphaera strain NCIMB 43385 modulates IL-6 and CD11bexpression in the brain of BALB/c mice.

FIG. 69 : NCIMB 42787 modulates TLR4 expression in the amygdala ofBALB/c mice.

DISCLOSURE OF THE INVENTION

Bacterial Strains

The compositions of the invention comprise a bacterial strain of thegenus Megasphaera. The Examples demonstrate that bacteria of this genusare useful for stimulating the immune system and for treating disease,in particular cancer. The preferred bacterial strains are of the speciesMegasphaera massiliensis.

Examples of Megasphaera species for use in the invention includeMegasphaera elsdenii, Megasphaera cerevisiae, Megasphaera massiliensis,Megasphaera indica, Megasphaera paucivorans, Megasphaera sueciensis andMegasphaera micronuciformis. A further example of a Megasphaera speciesfor use in the invention is Megasphaera hexanoica. The Megasphaera areobligately anaerobic, lactate-fermenting, gastrointestinal microbe ofruminant and non-ruminant mammals, including humans.

The type strain of M. massiliensis is NP3 (=CSUR P245=DSM 26228) [16].The GenBank accession number for the 16S rRNA gene sequences of M.massiliensis strain NP3 is JX424772.1.

All microorganism deposits were made under the terms of the BudapestTreaty and thus viability of the deposit is assured. Maintenance of aviable culture is assured for 30 years from the date of deposit. Duringthe pendency of the application, access to the deposit will be affordedto one determined by the Commissioner of the United States Patent andTrademark Office to be entitled thereto. All restrictions on theavailability to the public of the deposited microorganisms will beirrevocably removed upon the granting of a patent for this application.The deposit will be maintained for a term of at least thirty (30) yearsfrom the date of the deposit or for the enforceable life of the patentor for a period of at least five (5) years after the most recent requestfor the furnishing of a sample of the deposited material, whichever islongest. The deposit will be replaced should it become necessary due toinviability, contamination or loss of capability to function in themanner described in the specification.

The Megasphaera massiliensis bacterium tested in the Examples isreferred to herein as strain MRx0029. A 16S rRNA sequence for theMRx0029 strain that was tested is provided in SEQ ID NO: 1.

Strain MRx0029 was deposited with the international depositary authorityNCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) by 4DPharma Research Ltd. (Life Sciences Innovation Building, Cornhill Road,Aberdeen, AB25 2ZS, Scotland) on 13 Jul. 2017 as “Megasphaeramassiliensis MRx0029” and was assigned accession number NCIMB 42787.

Bacterial strains closely related to the strain tested in the Examplesare also expected to be effective for stimulating the immune system andfor treating and preventing disease, in particular cancer. In certainembodiments, the bacterial strain for use in the invention has a 16SrRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%identical to SEQ ID NO: 1. Preferably, the bacterial strain for use inthe invention has the 16S rRNA sequence represented by SEQ ID NO:1.

Bacterial strains that are biotypes of strains MRx0029 are also expectedto be effective for stimulating the immune system and for treating andpreventing disease, in particular cancer. A biotype is a closely relatedstrain that has the same or very similar physiological and biochemicalcharacteristics.

Strains that are biotypes of strains MRx0029 and that are suitable foruse in the invention may be identified by sequencing other nucleotidesequences for strains MRx0029. For example, substantially the wholegenome may be sequenced and a biotype strain for use in the inventionmay have at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequenceidentity across at least 80% of its whole genome (e.g. across at least85%, 90%, 95% or 99%, or across its whole genome). Other suitablesequences for use in identifying biotype strains may include hsp60 orrepetitive sequences such as BOX, ERIC, (GTG)₅ (SEQ ID NO: 15), or REPor [17]. Biotype strains may have sequences with at least 95%, 96%, 97%,98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequenceof the strains MRx0029.

Alternatively, strains that are biotypes of strains MRx0029 and that aresuitable for use in the invention may be identified by using strainsMRx0029 and restriction fragment analysis and/or PCR analysis, forexample by using fluorescent amplified fragment length polymorphism(FAFLP) and repetitive DNA element (rep)-PCR fingerprinting, or proteinprofiling, or partial 16S or 23S rDNA sequencing. In preferredembodiments, such techniques may be used to identify other Megasphaeramassiliensis strains.

In certain embodiments, strains that are biotypes of strains MRx0029 andthat are suitable for use in the invention are strains that provide thesame pattern as strains MRx0029 when analysed by amplified ribosomal DNArestriction analysis (ARDRA), for example when using Sau3AI restrictionenzyme (for exemplary methods and guidance see, for example, [18]).Alternatively, biotype strains are identified as strains that have thesame carbohydrate fermentation patterns as strains MRx0029.

Other Megasphaera strains that are useful in the compositions andmethods of the invention, such as biotypes of strains MRx0029, may beidentified using any appropriate method or strategy, including theassays described in the Examples. For instance, strains for use in theinvention may be identified by adding to cell lysate or whole cells andtesting for MAP2 expression, DRD2 expression, cytokine levels or cellsurvival. In particular, bacterial strains that have similar growthpatterns, metabolic type and/or surface antigens to strains MRx0029 maybe useful in the invention. A useful strain will have comparable immunemodulatory activity to strains MRx0029. In particular, a biotype strainwill elicit comparable effects on MAP2 expression, DRD2 expression,cytokine levels or cell survival as shown in the Examples, which may beidentified by using the culturing and administration protocols describedin the Examples. A biotype strain may elicit comparable effects onhistone deacetylase inhibitory activity as shown in the Examples, whichmay be identified by using the culturing and administration protocolsdescribed in the Examples.

In some embodiments, bacterial strains useful in the invention may beidentified by routinely profiling the production and consumption ofmetabolites by a bacterial strain. The inventors have found that thebacterial strain used in the Examples produces butyrate, valeric acidand hexanoic acid and consumes acetate and propionate (see FIGS. 54-56). The Megasphaera massiliensis strains Ref 1, Ref 2 and Ref 3 were alsofound to consume and produce these metabolites (see FIGS. 54-56 ).Therefore, in some embodiments, the bacterial strain of the inventionproduces one or more of the metabolites butyrate, valeric acid andhexanoic acid. In some embodiments, the bacterial strain of theinvention consumes one or both of acetate and propionate. In preferredembodiments, the bacterial strain of the invention produces butyrate,valeric acid and hexanoic acid and consumes acetate and propionate.

A particularly preferred strain of the invention is the Megasphaeramassiliensis MRx0029 strain. This is the exemplary strain tested in theExamples and shown to be effective for treating disease. Therefore, theinvention provides a cell, such as an isolated cell, of the Megasphaeramassiliensis strain MRx0029, or a derivative thereof. The invention alsoprovides a composition comprising a cell of the Megasphaera massiliensisstrain MRx0029, or a derivative thereof. The invention also provides abiologically pure culture of the Megasphaera massiliensis strainMRx0029. The invention also provides a cell of the Megasphaeramassiliensis strain MRx0029, or a derivative thereof, for use intherapy, in particular for the diseases described herein.

A particularly preferred strain of the invention is the Megasphaeramassiliensis strain deposited under accession number NCIMB 42787. Thisis the exemplary MRx0029 strain tested in the Examples and shown to beeffective stimulating the immune system and for treating and preventingdisease, in particular cancer. Therefore, the invention provides a cell,such as an isolated cell, of the Megasphaera massiliensis straindeposited under accession number NCIMB 42787, or a derivative thereof.The invention also provides a composition comprising a cell of theMegasphaera massiliensis strain deposited under accession number NCIMB42787, or a derivative thereof. The invention also provides abiologically pure culture of the Megasphaera massiliensis straindeposited under accession number NCIMB 42787. The invention alsoprovides a cell of the Megasphaera massiliensis strain deposited underaccession number NCIMB 42787, or a derivative thereof, for use intherapy, in particular for the diseases described herein.

A derivative of the strain of the invention may be a daughter strain(progeny) or a strain cultured (subcloned) from the original. Aderivative of a strain of the invention may be modified, for example atthe genetic level, without ablating the biological activity. Inparticular, a derivative strain of the invention is therapeuticallyactive. A derivative strain will have comparable therapeutic activity tothe MRx0029 strain. In particular, a derivative strain will elicitcomparable effects on MAP2 expression, DRD2 expression, cytokine levelsor cell survival as shown in the Examples, which may be identified byusing the culturing and administration protocols described in theExamples. A derivative strain may elicit comparable effects on histonedeacetylase inhibitory activity as shown in the Examples, which may beidentified by using the culturing and administration protocols describedin the Examples. A derivative of the MRx0029 strain will generally be abiotype of the MRx0029 strain.

References to cells of the Megasphaera massiliensis MRx0029 strainencompass any cells that have the same safety and therapeutic efficacycharacteristics as the strain MRx0029, and such cells are encompassed bythe invention.

In preferred embodiments, the bacterial strains in the compositions ofthe invention are viable and capable of partially or totally colonisingthe intestine.

The inventors have found that Megasphaera massiliensis strains increasethe activation of inflammatory cytokines such as IL-1β, TNF-α, MIP-3α,IL-23, IL-8 and/or IL-6.

The inventors have found that Megasphaera massiliensis strains increasethe activation of immune cells and enhance secretion of cytokines suchas IL-1β, TNF-α, MIP-3α, IL-23, IL-8 and/or IL-6.

In preferred embodiments, the invention provides a compositioncomprising the strain deposited at NCIMB under accession number NCIMB42787, or a derivative or biotype thereof, preferably for use instimulating the immune system and for treating and preventing disease,in particular cancer, most preferably brain cancer, such asneuroblastoma. In preferred embodiments, the invention provides acomposition comprising the strain deposited at NCIMB under accessionnumber NCIMB 42787, or a derivative or biotype thereof, preferably foruse in treating or preventing metastatic melanoma, breast cancer,ovarian cancer, cervical cancer, glioblastoma, carcinoma, lung cancer,chronic lymphocyte leukemia, prostate cancer, lymphoma, gastric cancer,colorectal cancer and/or haematological malignancies.

In preferred embodiments, the bacterial strains in the compositions ofthe invention are viable and capable of partially or totally colonisingthe intestine.

In certain embodiments, the composition of the invention does notcomprise a cell of the Megasphaera massiliensis strain 42787.

In some embodiments, the bacterial strain in the compositions of theinvention is a bacterial strain of the genus Megasphaera, wherein thebacterial strain is not the strain deposited under accession numberNCIMB 42787.

In some embodiments, the bacterial strain in the compositions of theinvention is a bacterial strain of the species Megasphaera massiliensis,wherein the bacterial strain is not the strain deposited under accessionnumber NCIMB 42787.

These bacterial strains were deposited with the international depositaryauthority NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland)by 4D Pharma Research Ltd. (Life Sciences Innovation Building, CornhillRoad, Aberdeen, AB25 2ZS, Scotland) on 6 May 2019 as Megasphaeramassiliensis (under accession numbers NCIMB 43388 and NCIMB 43389) andMegasphaera spp. (accession numbers NCIMB 43385, NCIMB 43386 and NCIMB43387). Accordingly, in an alternative embodiment, the compositions ofthe invention comprise one or more of these bacterial strains, orbiotypes or derivatives thereof. For the avoidance of doubt, Ref 1referred to above is the strain deposited under accession number NCIMB43385, Ref 2 referred to above is the strain deposited under accessionnumber NCIMB 43388, and Ref 3 referred to above is the strain depositedunder accession number NCIMB 43389. All microorganism deposits were madeunder the terms of the Budapest Treaty and thus viability of the depositis assured. Maintenance of a viable culture is assured for 30 years fromthe date of deposit. During the pendency of the application, access tothe deposit will be afforded to one determined by the Commissioner ofthe United States Patent and Trademark Office to be entitled thereto.All restrictions on the availability to the public of the depositedmicroorganisms will be irrevocably removed upon the granting of a patentfor this application. The deposit will be maintained for a term of atleast thirty (30) years from the date of the deposit or for theenforceable life of the patent or for a period of at least five (5)years after the most recent request for the furnishing of a sample ofthe deposited material, whichever is longest. The deposit will bereplaced should it become necessary due to inviability, contamination orloss of capability to function in the manner described in thespecification.

Bacterial strains closely related to the strains tested in the Examplesare also expected to be effective for stimulating the immune system andfor treating and preventing disease, in particular cancer.

In certain embodiments, the bacterial strain for use in the invention isthe Megasphaera massiliensis strain deposited under accession numberNCIMB 43388. In certain embodiments, the invention provides a cell ofthe strain deposited under accession number NCIMB 43388, or a derivativethereof, for use in therapy. In certain embodiments, the inventionprovides a cell of the strain deposited under accession number NCIMB43388, or derivative thereof for use in stimulating the immune systemand for treating and preventing disease, in particular cancer. Incertain embodiments, the invention provides a cell of the straindeposited under accession number NCIMB 43388, for use in any one of thediseases described herein.

In preferred embodiments, the invention provides a compositioncomprising the strain deposited at NCIMB under accession number NCIMB43388, or a derivative or biotype thereof, preferably for use instimulating the immune system and for treating and preventing disease,in particular cancer, most preferably brain cancer, such asneuroblastoma. In preferred embodiments, the invention provides acomposition comprising the strain deposited at NCIMB under accessionnumber NCIMB 43388, or a derivative or biotype thereof, preferably foruse in treating or preventing metastatic melanoma, breast cancer,ovarian cancer, cervical cancer, glioblastoma, carcinoma, lung cancer,chronic lymphocyte leukemia, prostate cancer, lymphoma, gastric cancer,colorectal cancer and/or haematological malignancies.

In certain embodiments, the composition of the invention does notcomprise a cell of the Megasphaera massiliensis strain deposited underaccession number NCIMB 43388. In some embodiments, the bacterial strainin the compositions of the invention is a bacterial strain of the genusMegasphaera, wherein the bacterial strain is not the strain depositedunder accession number NCIMB 43388. In some embodiments, the bacterialstrain in the compositions of the invention is a bacterial strain of thespecies Megasphaera massiliensis, wherein the bacterial strain is notthe strain deposited under accession number NCIMB 43388.

Accordingly, in certain embodiments, the bacterial strain for use in theinvention is the Megasphaera massiliensis deposited under accessionnumber NCIMB 43389. In certain embodiments, the invention provides acell of the strain deposited under accession number NCIMB 43389, or aderivative thereof, for use in therapy. In certain embodiments, theinvention provides a cell of the strain deposited under accession numberNCIMB 43389, or derivative thereof for use in stimulating the immunesystem and for treating and preventing disease, in particular cancer. Incertain embodiments, the invention provides a cell of the straindeposited under accession number NCIMB 43389, for use in any one of thediseases described herein.

In preferred embodiments, the invention provides a compositioncomprising the strain deposited at NCIMB under accession number NCIMB43389, or a derivative or biotype thereof, preferably for use instimulating the immune system and for treating and preventing disease,in particular cancer, most preferably brain cancer, such asneuroblastoma. In preferred embodiments, the invention provides acomposition comprising the strain deposited at NCIMB under accessionnumber NCIMB 43389, or a derivative or biotype thereof, preferably foruse in treating or preventing metastatic melanoma, breast cancer,ovarian cancer, cervical cancer, glioblastoma, carcinoma, lung cancer,chronic lymphocyte leukemia, prostate cancer, lymphoma, gastric cancer,colorectal cancer and/or haematological malignancies.

In certain embodiments, the composition of the invention does notcomprise a cell of the Megasphaera massiliensis strain deposited underaccession number NCIMB 43389. In some embodiments, the bacterial strainin the compositions of the invention is a bacterial strain of the genusMegasphaera, wherein the bacterial strain is not the strain depositedunder accession number NCIMB 43389. In some embodiments, the bacterialstrain in the compositions of the invention is a bacterial strain of thespecies Megasphaera massiliensis, wherein the bacterial strain is notthe strain deposited under accession number NCIMB 43389.

In certain embodiments, the bacterial strain for use in the inventionhas a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%or 99.9% identical to SEQ ID NO:9. In certain embodiments, the bacterialstrain for use in the invention has the 16S rRNA sequence represented bySEQ ID NO:9. In certain embodiments, the invention provides a bacterialstrain having a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%,99%, 99.5% or 99.9% identical to SEQ ID NO:9 for use in therapy. Incertain embodiments, the invention provides a bacterial strain havingthe 16S rRNA sequence represented by SEQ ID NO:9 for use in therapy.

In certain embodiments, the bacterial strain for use in the inventionhas a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%or 99.9% identical to SEQ ID NO:10. In certain embodiments, thebacterial strain for use in the invention has the 16S rRNA sequencerepresented by SEQ ID NO: 10. In certain embodiments, the inventionprovides a bacterial strain having a 16S rRNA sequence that is at least95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:10 foruse in therapy. In certain embodiments, the invention provides abacterial strain having the 16S rRNA sequence represented by SEQ IDNO:10 for use in therapy.

In certain embodiments, the bacterial strain for use in the inventionhas a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%or 99.9% identical to the 16S rRNA sequence of a bacterial strain of thegenus Megasphaera. In certain embodiments, the bacterial strain for usein the invention is of the genus Megasphaera.

In certain embodiments, the bacterial strain for use in the invention isthe Megasphaera strain deposited under accession number NCIMB 43385. Incertain embodiments, the invention provides a cell of the straindeposited under accession number NCIMB 43385, or a derivative thereof,for use in therapy. In certain embodiments, the invention provides acell of the strain deposited under accession number NCIMB 43385, orderivative thereof for use in stimulating the immune system and fortreating and preventing disease, in particular cancer. In certainembodiments, the invention provides a cell of the strain deposited underaccession number NCIMB 43385, for use in any one of the diseasesdescribed herein.

In preferred embodiments, the invention provides a compositioncomprising the strain deposited at NCIMB under accession number NCIMB43385, or a derivative or biotype thereof, preferably for use instimulating the immune system and for treating and preventing disease,in particular cancer, most preferably brain cancer, such asneuroblastoma. In preferred embodiments, the invention provides acomposition comprising the strain deposited at NCIMB under accessionnumber NCIMB 43385, or a derivative or biotype thereof, preferably foruse in treating or preventing metastatic melanoma, breast cancer,ovarian cancer, cervical cancer, glioblastoma, carcinoma, lung cancer,chronic lymphocyte leukemia, prostate cancer, lymphoma, gastric cancer,colorectal cancer and/or haematological malignancies.

In certain embodiments, the composition of the invention does notcomprise a cell of the Megasphaera massiliensis strain deposited underaccession number NCIMB 43385. In some embodiments, the bacterial strainin the compositions of the invention is a bacterial strain of the genusMegasphaera, wherein the bacterial strain is not the strain depositedunder accession number NCIMB 43385. In some embodiments, the bacterialstrain in the compositions of the invention is a bacterial strain of thespecies Megasphaera massiliensis, wherein the bacterial strain is notthe strain deposited under accession number NCIMB 43385.

In certain embodiments, the bacterial strain for use in the invention isthe Megasphaera strain deposited under accession number NCIMB 43386. Incertain embodiments, the invention provides a cell of the straindeposited under accession number NCIMB 43386, or a derivative thereof,for use in therapy. In certain embodiments, the invention provides acell of the strain deposited under accession number NCIMB 43386, orderivative thereof for use in stimulating the immune system and fortreating and preventing disease, in particular cancer. In certainembodiments, the invention provides a cell of the strain deposited underaccession number NCIMB 43386, for use in any one of the diseasesdescribed herein.

In preferred embodiments, the invention provides a compositioncomprising the strain deposited at NCIMB under accession number NCIMB43386, or a derivative or biotype thereof, preferably for use instimulating the immune system and for treating and preventing disease,in particular cancer, most preferably brain cancer, such asneuroblastoma. In preferred embodiments, the invention provides acomposition comprising the strain deposited at NCIMB under accessionnumber NCIMB 43386, or a derivative or biotype thereof, preferably foruse in treating or preventing metastatic melanoma, breast cancer,ovarian cancer, cervical cancer, glioblastoma, carcinoma, lung cancer,chronic lymphocyte leukemia, prostate cancer, lymphoma, gastric cancer,colorectal cancer and/or haematological malignancies.

In certain embodiments, the composition of the invention does notcomprise a cell of the Megasphaera massiliensis strain deposited underaccession number NCIMB 43386. In some embodiments, the bacterial strainin the compositions of the invention is a bacterial strain of the genusMegasphaera, wherein the bacterial strain is not the strain depositedunder accession number NCIMB 43386. In some embodiments, the bacterialstrain in the compositions of the invention is a bacterial strain of thespecies Megasphaera massiliensis, wherein the bacterial strain is notthe strain deposited under accession number NCIMB 43386.

In certain embodiments, the bacterial strain for use in the invention isthe Megasphaera strain deposited under accession number NCIMB 43387. Incertain embodiments, the invention provides a cell of the straindeposited under accession number NCIMB 43387, or a derivative thereof,for use in therapy. In certain embodiments, the invention provides acell of the strain deposited under accession number NCIMB 43387, orderivative thereof for use in stimulating the immune system and fortreating and preventing disease, in particular cancer. In certainembodiments, the invention provides a cell of the strain deposited underaccession number NCIMB 43387, for use in any one of the diseasesdescribed herein.

In preferred embodiments, the invention provides a compositioncomprising the strain deposited at NCIMB under accession number NCIMB43387, or a derivative or biotype thereof, preferably for use instimulating the immune system and for treating and preventing disease,in particular cancer, most preferably brain cancer, such asneuroblastoma. In preferred embodiments, the invention provides acomposition comprising the strain deposited at NCIMB under accessionnumber NCIMB 43387, or a derivative or biotype thereof, preferably foruse in treating or preventing metastatic melanoma, breast cancer,ovarian cancer, cervical cancer, glioblastoma, carcinoma, lung cancer,chronic lymphocyte leukemia, prostate cancer, lymphoma, gastric cancer,colorectal cancer and/or haematological malignancies.

In certain embodiments, the composition of the invention does notcomprise a cell of the Megasphaera massiliensis strain deposited underaccession number NCIMB 43387. In some embodiments, the bacterial strainin the compositions of the invention is a bacterial strain of the genusMegasphaera, wherein the bacterial strain is not the strain depositedunder accession number NCIMB 43387. In some embodiments, the bacterialstrain in the compositions of the invention is a bacterial strain of thespecies Megasphaera massiliensis, wherein the bacterial strain is notthe strain deposited under accession number NCIMB 43387.

In certain embodiments, the bacterial strain for use in the inventionhas a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%or 99.9% identical to SEQ ID NO:8. In certain embodiments, the bacterialstrain for use in the invention has a 16S rRNA sequence that is at least95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO: 11. Incertain embodiments, the bacterial strain for use in the invention has a16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or99.9% identical to SEQ ID NO: 12. In certain embodiments, the bacterialstrain for use in the invention has a 16S rRNA sequence that is at least95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NOs:8, 11 or12. In certain embodiments, the invention provides a bacterial strainhaving a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%,99.5% or 99.9% identical to SEQ ID NOs:8, 11 or 12 for use in therapy.

In certain embodiments, the bacterial strain for use in the inventionhas the 16S rRNA sequence represented by SEQ ID NO:8. In certainembodiments, the bacterial strain for use in the invention has the 16SrRNA sequence represented by SEQ ID NO: 11. In certain embodiments, thebacterial strain for use in the invention has the 16S rRNA sequencerepresented by SEQ ID NO: 12. In certain embodiments, the bacterialstrain for use in the invention has the 16S rRNA sequence represented bySEQ ID NOs: 8, 11 or 12. In certain embodiments, the invention providesa bacterial strain having the 16S rRNA sequence represented by SEQ IDNOs: 8, 11 or 12 for use in therapy.

Bacterial strains that are biotypes of one or more of the strainsdeposited under accession numbers NCIMB 43385, NCIMB 43386, NCIMB 43387,NCIMB 43388 and/or NCIMB 43389 are also expected to be effective forstimulating the immune system and for treating and preventing disease,in particular cancer. A biotype is a closely related strain that has thesame or very similar physiological and biochemical characteristics.

In certain embodiments, the invention provides the bacterial strainsdeposited under accession numbers NCIMB 43385, NCIMB 43386, NCIMB 43387,NCIMB 43388 and/or NCIMB 43389, or biotypes thereof, for use in therapy.

Strains that are biotypes of one or more of the strains deposited underaccession numbers NCIMB 43385, NCIMB 43386, NCIMB 43387, NCIMB 43388and/or NCIMB 43389 and that are suitable for use in the invention may beidentified by sequencing other nucleotide sequences for one or more ofthe strains deposited under accession numbers NCIMB 43385, NCIMB 43386,NCIMB 43387, NCIMB 43388 and/or NCIMB 43389. For example, substantiallythe whole genome may be sequenced and a biotype strain for use in theinvention may have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or99.9% sequence identity across at least 80% of its whole genome (e.g.across at least 85%, 90%, 95% or 99%, or across its whole genome). Othersuitable sequences for use in identifying biotype strains may includehsp60 or repetitive sequences such as BOX, ERIC, (GTG)₅ (SEQ ID NO: 15),or REP. Biotype strains may have sequences with at least 85%, 90%, 95%,96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to thecorresponding sequence of one or more of the strains deposited underaccession numbers NCIMB 43385, NCIMB 43386, NCIMB 43387, NCIMB 43388and/or NCIMB 43389.

Alternatively, strains that are biotypes of one or more of the strainsdeposited under accession numbers NCIMB 43385, NCIMB 43386, NCIMB 43387,NCIMB 43388 and/or NCIMB 43389 and that are suitable for use in theinvention may be identified by using one or more of the strainsdeposited under accession numbers NCIMB 43385, NCIMB 43386, NCIMB 43387,NCIMB 43388 and/or NCIMB 43389 and restriction fragment analysis and/orPCR analysis, for example by using fluorescent amplified fragment lengthpolymorphism (FAFLP) and repetitive DNA element (rep)-PCRfingerprinting, or protein profiling, or partial 16S or 23S rDNAsequencing. In preferred embodiments, such techniques may be used toidentify other Megasphaera massiliensis strains.

In certain embodiments, strains that are biotypes of one or more of thestrains deposited under accession numbers NCIMB 43385, NCIMB 43386,NCIMB 43387, NCIMB 43388 and/or NCIMB 43389 and that are suitable foruse in the invention are strains that provide the same pattern as one ormore of the strains deposited under accession numbers NCIMB 43385, NCIMB43386, NCIMB 43387, NCIMB 43388 and/or NCIMB 43389 when analysed byamplified ribosomal DNA restriction analysis (ARDRA), for example whenusing Sau3AI restriction enzyme. Alternatively, biotype strains areidentified as strains that have the same carbohydrate fermentationpatterns as one or more of the strains deposited under accession numbersNCIMB 43385, NCIMB 43386, NCIMB 43387, NCIMB 43388 and/or NCIMB 43389.

Other strains that are useful in the compositions and methods of theinvention, such as biotypes of one or more of the strains depositedunder accession numbers NCIMB 43385, NCIMB 43386, NCIMB 43387, NCIMB43388 and/or NCIMB 43389, may be identified using any appropriate methodor strategy, including the assays described in the Examples. Forinstance, strains for use in the invention may be identified by addingto cell lysate or whole cells and testing for MAP2 expression, DRD2expression, cytokine levels or cell survival. In particular, bacterialstrains that have similar growth patterns, metabolic type and/or surfaceantigens to one or more of the strains deposited under accession numbersNCIMB 43385, NCIMB 43386, NCIMB 43387, NCIMB 43388 and/or NCIMB 43389may be useful in the invention. A useful strain will have comparableimmune modulatory activity to one or more of the strains deposited underaccession numbers NCIMB 43385, NCIMB 43386, NCIMB 43387, NCIMB 43388and/or NCIMB 43389. In particular, a biotype strain will elicitcomparable effects on MAP2 expression, DRD2 expression, cytokine levelsor cell survival as shown in the Examples, which may be identified byusing the culturing and administration protocols described in theExamples. A biotype strain may elicit comparable effects on histonedeacetylase inhibitory activity as shown in the Examples, which may beidentified by using the culturing and administration protocols describedin the Examples.

In certain embodiments, preferred strains of the invention are strainsdeposited under accession numbers NCIMB 43385, NCIMB 43386, NCIMB 43387,NCIMB 43388 and/or NCIMB 43389. These are exemplary strains tested inthe Examples and shown to be effective for treating disease. Therefore,the invention provides a cell, such as an isolated cell, of one or moreof the strains deposited under accession numbers NCIMB 43385, NCIMB43386, NCIMB 43387, NCIMB 43388 and/or NCIMB 43389, or a derivativethereof. The invention also provides a composition comprising a cell ofone of more of the strains deposited under accession numbers NCIMB43385, NCIMB 43386, NCIMB 43387, NCIMB 43388 and/or NCIMB 43389, or aderivative thereof. The invention also provides a biologically pureculture of one or more of the strains deposited under accession numbersNCIMB 43385, NCIMB 43386, NCIMB 43387, NCIMB 43388 and/or NCIMB 43389.The invention also provides a cell of one or more of the strainsdeposited under accession numbers NCIMB 43385, NCIMB 43386, NCIMB 43387,NCIMB 43388 and/or NCIMB 43389, or a derivative thereof, for use intherapy, in particular for the diseases described herein.

A derivative of the strain of the invention may be a daughter strain(progeny) or a strain cultured (subcloned) from the original. Aderivative of a strain of the invention may be modified, for example atthe genetic level, without ablating the biological activity. Inparticular, a derivative strain of the invention is therapeuticallyactive. A derivative strain will have comparable therapeutic activity toone or more of the strains deposited under accession numbers NCIMB43385, NCIMB 43386, NCIMB 43387, NCIMB 43388 and/or NCIMB 43389. Inparticular, a derivative strain will elicit comparable effects on MAP2expression, DRD2 expression, cytokine levels or cell survival as shownin the Examples, which may be identified by using the culturing andadministration protocols described in the Examples. A derivative strainmay elicit comparable effects on histone deacetylase inhibitory activityas shown in the Examples, which may be identified by using the culturingand administration protocols described in the Examples. A derivative ofone or more of the strains deposited under accession numbers NCIMB43385, NCIMB 43386, NCIMB 43387, NCIMB 43388 and/or NCIMB 43389 willgenerally be a biotype of one or more of the strains deposited underaccession numbers NCIMB 43385, NCIMB 43386, NCIMB 43387, NCIMB 43388and/or NCIMB 43389, respectively.

The inventors have found that the bacterial strain used in the Examplesproduces 2-methyl-propanoic acid and 3-methyl-butanoic acid and consumesformic acid (see FIG. 58 ). The strains deposited under accessionnumbers NCIMB 43385, NCIMB 43388 and NCIMB 43389 were also found toproduce 2-methyl-propanoic acid and 3-methyl-butanoic acid. In addition,the strains deposited under accession numbers NCIMB 43385 and NCIMB43388 were also found to consume formic acid. Therefore, in someembodiments, the bacterial strain of the invention produces one or moreof the metabolites 2-methyl-propanoic acid and—3-methyl-butanoic acid.In some embodiments, the bacterial strain of the invention consumesformic acid. In some embodiments, the bacterial strain of the inventionproduces 2-methyl-propanoic acid and 3-methyl-butanoic acid and consumesformic acid. In preferred embodiments, the bacterial strain of theinvention produces butyrate, valeric acid, hexanoic acid,2-methyl-propanoic acid and—3-methyl-butanoic acid, and consumesacetate, propionate and formic acid.

In certain embodiments, the production of butyrate and/or valeric acidgenerates IL-8 secretion. Accordingly, in certain embodiments, thecompositions of the invention may stimulate the immune system via theproduction of butyrate and/or valeric acid.

In certain embodiments, the compositions of the invention do notcomprise Megasphaera elsdenii. In certain embodiments, the bacterialstrain useful in the compositions and methods of the invention is notMegasphaera elsdenii.

Therapeutic Uses

Stimulating the Immune System

The Examples show that administration of the compositions of theinvention can lead to immune stimulation in human peripheral bloodmononuclear cells (PBMCs). Since administration of the compositions ofthe invention were shown to have an immunostimulatory effect on PBMCs,compositions of the invention may be useful in the treatment of disease,in particular diseases characterised by reduced immune activation anddiseases treatable by an increased immune response. In certainembodiments, the compositions of the invention are for use instimulating the immune system. In certain embodiments, the compositionsof the invention are for use in treating disease by stimulating theimmune system. In certain embodiments, the compositions of the inventionare for use in promoting an immune response.

The Examples show that administration of the compositions of theinvention can lead to a decrease in the percentage of Tregs in PBMCs(FIG. 6C). Tregs, also known as suppressor T cells, are a population ofT cells which function to suppress the immune response. Tregs arecharacterised by the high expression of cell-surface marker CD25 and thelow expression of CD127 [19]. Since administration of the compositionsof the invention were shown to selectively reduce the population ofTregs (FIG. 6C), compositions of the invention may be useful in thetreatment of diseases characterised by an increase in the percentage ofTregs in a cell population. In one embodiment, the compositions of theinvention may be useful for treating or preventing diseasescharacterised by an increase in the percentage of Tregs in a cellpopulation. In one embodiment, the compositions of the invention may beuseful for treating or preventing diseases characterised by an increasein the percentage of CD4+CD25+CD127− cells in a cell population. In oneembodiment, the compositions of the invention are for use in treating orpreventing diseases by decreasing the percentage of Tregs in cellpopulations. In one embodiment, compositions of the invention are foruse in reducing suppression of the immune response by Tregs. In oneembodiment, compositions of the invention are for use in stimulating theimmune response by the selective reduction of Tregs. In one embodiment,compositions of the invention are for use in immunostimulation, whereinthe compositions of the invention reduce the number or percentage ofTregs.

The Examples demonstrate that the compositions of the invention may beable to selectively target Tregs, without significantly affecting cellssuch as B cells, CD4 T cells or CD8 T cells. Therefore, compositions ofthe invention may selectively reduce Tregs in PBMCs, withoutsignificantly affecting the percentage of the other cell types tested.In one embodiment, compositions of the invention are for use inselectively reducing the number or percentage of Tregs, wherein thenumber or percentage of CD4 T cells does not significantly change. Inone embodiment, compositions of the invention are for use in selectivelyreducing the number or percentage of Tregs, wherein the number orpercentage of CD8 T cells does not significantly change. In oneembodiment, compositions of the invention are for use in selectivelyreducing the number or percentage of Tregs, wherein the number orpercentage of B cells does not significantly change. In a furtherembodiment, compositions of the invention are for use in selectivelyreducing the number or percentage of Tregs, wherein the number orpercentage of B cells, CD4 T cells and/or CD8 T cells does notsignificantly change.

The decrease in the percentage of Tregs was particularly surprisingbecause the Megasphaera massiliensis MRx0029 strain produces butyrate,and butyrate has been associated with increased Treg cell levels in theblood and increased Treg activity [20]. Therefore, it was unexpectedthat the compositions of the invention would lead to a decrease in thepercentage of Tregs in PBMCs.

The Examples also show that administration of the compositions of theinvention can lead to an increase in the ratio of CD8 cells to Tregcells. CD8+ T cells (CD8 cells) are cytotoxic T cells, and play keyroles in the immune defence against intracellular pathogens. Sinceadministration of the compositions of the invention were shown toincrease the ratio of both CD8/Treg cells and activated CD8/Treg cells(FIG. 6G and FIG. 6H), compositions of the invention may be useful inthe treatment of diseases characterised by a decrease in the ratio ofCD8/Treg and/or activated CD8/Treg cells. In one embodiment, thecompositions of the invention are for use in treating or preventingdiseases characterised by decrease in the ratio of CD8/Treg cells. Inone embodiment, the compositions of the invention are for use intreating or preventing diseases characterised by decrease in the ratioof activated CD8/Treg cells. In one embodiment, the compositions of theinvention are for use in treating or preventing diseases by decreasingthe percentage of Tregs in cell populations, thereby increasing theratio of CD8/Treg cells. In one embodiment, the compositions of theinvention are for use in treating or preventing diseases by decreasingthe percentage of Tregs in cell populations, thereby increasing theratio of CD8/Treg cells, wherein the increase in the ratio of CD8/Tregcells results in immunostimulation. In another embodiment, thecompositions of the invention are for use in treating or preventingdiseases by decreasing the percentage of Tregs in cell populations,thereby increasing the ratio of activated CD8/Treg cells. In oneembodiment, the compositions of the invention are for use in treating orpreventing diseases by decreasing the percentage of Tregs in cellpopulations, thereby increasing the ratio of CD8/Treg cells, wherein theincrease in the ratio of activated CD8/Treg cells results in immunestimulation. In one embodiment, compositions of the invention are foruse in stimulating the immune response by increasing the ratio ofCD8/Treg cells. In one embodiment, compositions of the invention are foruse in stimulating the immune response by increasing the ratio ofactivated CD8/Treg cells.

The Examples also show that administration of the compositions of theinvention can lead to an increase in the percentage of CD19+CD3− cellsin PBMCs (FIG. 6F). Therefore, administration of the compositions of theinvention can lead to an increase in the percentage of B cells in a cellpopulation. Since administration of the compositions of the inventionwere shown to increase the percentage of B cells, compositions of theinvention may be useful in the treatment of diseases characterised by adecrease in the number or percentage of B cells. In one embodiment, thecompositions of the invention are for use in treating or preventingdiseases characterised by decrease in the number or percentage of Bcells. In one embodiment, the compositions of the invention are for usein treating or preventing diseases characterised by decrease in thenumber or percentage of CD19+CD3− cells. In one embodiment, thecompositions of the invention are for use in treating or preventingdiseases by increasing the number or percentage of B cells in cellpopulations, wherein the increase in number or percentage of B cellsresults in immune stimulation. In one embodiment, compositions of theinvention are for use in stimulating the immune response by increasingthe number or percentage of B cells.

The Examples also show that administration of the compositions of theinvention can lead to an increase in the percentage of CD8 T-cytotoxiccells (FIG. 6D) in PBMCs. Therefore, administration of the compositionsof the invention can lead to an increase in the percentage of CD8 Tcells in a cell population. Since administration of the compositions ofthe invention were shown to increase the percentage of CD8 T-cytotoxiccells, compositions of the invention may be useful in the treatment ofdiseases characterised by a decrease in the number or percentage of CD8T-cytotoxic cells. In one embodiment, the compositions of the inventionare for use in treating or preventing diseases characterised by decreasein the number or percentage of CD8 T-cytotoxic cells. In one embodiment,the compositions of the invention are for use in treating or preventingdiseases by increasing the number or percentage of CD8 T-cytotoxic cellsin cell populations, wherein the increase in number or percentage of CD8T-cytotoxic cells results in immune stimulation. In one embodiment,compositions of the invention are for use in stimulating the immuneresponse by increasing the number or percentage of CD8 T-cytotoxiccells.

The Examples also show that administration of the compositions of theinvention can lead to an increase in the percentage of CD8+ activatedcells (FIG. 6E) in PBMCs. Therefore, administration of the compositionsof the invention can lead to an increase in the percentage of CD8+activated cells in a cell population. Since administration of thecompositions of the invention were shown to increase the percentage ofCD8+ activated cells, compositions of the invention may be useful in thetreatment of diseases characterised by a decrease in the number orpercentage of CD8+ activated cells. In one embodiment, the compositionsof the invention are for use in treating or preventing diseasescharacterised by decrease in the number or percentage of CD8+ activatedcells. In one embodiment, the compositions of the invention are for usein treating or preventing diseases by increasing the number orpercentage of CD8+ activated cells in cell populations, wherein theincrease in number or percentage of CD8+ activated cells results inimmune stimulation. In one embodiment, compositions of the invention arefor use in stimulating the immune response by increasing the number orpercentage of CD8+ activated cells.

The Examples show that administration of the compositions of theinvention can lead to an increase in expression of pro-inflammatorymolecules in PBMCs, such as pro-inflammatory cytokines (FIG. 7 and FIG.9 ). Examples of immune-stimulatory (e.g. pro-inflammatory) moleculesthat showed an increase in expression levels upon administration ofcompositions of the invention include IL-23, TNF-α, IL-1β, MIP-3α, IL-8and IL-6. Since administration of the compositions of the invention wereshown to increase the expression of immune-stimulatory (e.g.pro-inflammatory) molecules, compositions of the invention may be usefulin the treatment of diseases characterised by a decrease in expressionof pro-inflammatory molecules, such as pro-inflammatory cytokines. Inone embodiment, the compositions of the invention are for use intreating or preventing diseases characterised by a decrease in theexpression and/or activity of pro-inflammatory molecules, in particulardiseases characterised by a decrease in the expression and/or activityof pro-inflammatory cytokines. In a particular embodiment, thecompositions of the invention are for use in treating or preventingdiseases characterised by a decrease in the expression and/or activityof IL-23, TNF-α, IL-1β, MIP-3α and/or IL-6. In a particular embodiment,the compositions of the invention are for use in treating or preventingdiseases characterised by a decrease in the expression and/or activityof IL-8. In a particular embodiment, the compositions of the inventionare for use in treating or preventing diseases characterised by adecrease in the expression and/or activity of CD11b. In one embodiment,the compositions of the invention are for use in treating or preventingdiseases by increasing the expression and/or activity of IL-23, TNF-α,IL-1β, MIP-3α and/or IL-6. In one embodiment, the compositions of theinvention are for use in treating or preventing diseases by increasingthe expression and/or activity of IL-8. In one embodiment, thecompositions of the invention are for use in treating or preventingdiseases by increasing the expression and/or activity of CD11b. In oneembodiment, compositions of the invention are for use in promoting theimmune response by increasing the expression and/or activity of IL-23,TNF-α, IL-1β, MIP-3α and/or IL-6. In one embodiment, compositions of theinvention are for use in promoting the immune response by increasing theexpression and/or activity of IL-8. In one embodiment, compositions ofthe invention are for use in promoting the immune response by increasingthe expression and/or activity of CD11b.

The Examples also show that administration of the compositions of theinvention can lead to an increase in expression of IL-1β in PBMCs. IL-1βis a pro-inflammatory cytokine [21]. The production and secretion ofIL-1β is regulated by the inflammasome, a protein complex which isassociated with activation of the inflammatory response [22]. Sinceadministration of the compositions of the invention were shown toincrease the expression of IL-1β, compositions of the invention may beuseful in the treatment of diseases characterised by a decrease inexpression of IL-1β. In a particular embodiment, the compositions of theinvention are for use in treating or preventing diseases characterisedby a decrease in the expression and/or activity of IL-1β. In oneembodiment, the compositions of the invention are for use in treating orpreventing diseases by increasing the expression and/or activity ofIL-1β. In one embodiment, the compositions of the invention are for usein promoting the immune response by increasing the expression and/oractivity of IL-1β. In certain embodiments, a bacterial strain of thespecies Megasphaera massiliensis is for use in the treatment of diseasescharacterised by a decrease in expression and/or activity of IL-1β. Inone embodiment, a bacterial strain of the species Megasphaeramassiliensis is for use in treating or preventing diseases by increasingthe expression and/or activity of IL-1β. In one embodiment, a bacterialstrain of the species Megasphaera massiliensis is for use in promotingthe immune response by increasing the expression and/or activity ofIL-1β.

The Examples also show that administration of the compositions of theinvention can lead to an increase in expression of IL-23. IL-23 has beenlinked to inflammation [23,24]. The proposed functions of IL-23 in theimmune response include promoting the proliferation of CD4+ memory Tcells and promoting the secretion of IFN-γ by dendritic cells (DCs)[25]. Since administration of the compositions of the invention wereshown to increase the expression of IL-23, compositions of the inventionmay be useful in the treatment of diseases characterised by a decreasein expression of IL-23. In a particular embodiment, the compositions ofthe invention are for use in treating or preventing diseasescharacterised by a decrease in the expression and/or activity of IL-23.In one embodiment, the compositions of the invention are for use intreating or preventing diseases by increasing the expression and/oractivity of IL-23. In one embodiment, compositions of the invention arefor use in promoting the immune response by increasing the expressionand/or activity of IL-23. In certain embodiments, a bacterial strain ofthe species Megasphaera massiliensis is for use in the treatment ofdiseases characterised by a decrease in expression and/or activity ofIL-23. In one embodiment, a bacterial strain of the species Megasphaeramassiliensis is for use in treating or preventing diseases by increasingthe expression and/or activity of IL-23. In one embodiment, a bacterialstrain of the species Megasphaera massiliensis is for use in promotingthe immune response by increasing the expression and/or activity ofIL-23.

The Examples also show that administration of the compositions of theinvention can lead to an increase in expression of MacrophageInflammatory Protein-3 (MIP3-α), or CCL20 in PBMCs. MIP3-α is aninflammatory chemokine which binds to the CCR6 receptor, and functionsas a chemoattractant for DCs and memory T-cells. MIP3-α is associatedwith triggering the adaptive immune response by recruiting immature DCsto the site of inflammation [26]. Dysregulated expression of MIP3-α hasbeen associated with diseases such as inflammatory bowel disease [27].Since administration of the compositions of the invention were shown toincrease the expression of MIP3-α, compositions of the invention may beuseful in the treatment of diseases characterised by a decrease inexpression of MIP3-α. In a particular embodiment, the compositions ofthe invention are for use in treating or preventing diseasescharacterised by a decrease in the expression and/or activity of MIP3-α.In one embodiment, the compositions of the invention are for use intreating or preventing diseases by increasing the expression and/oractivity of MIP3-α. In one embodiment, compositions of the invention arefor use in promoting the immune response by increasing the expressionand/or activity of MIP3-α. In certain embodiments, a bacterial strain ofthe species Megasphaera massiliensis is for use in the treatment ofdiseases characterised by a decrease in expression of and/or activity ofMIP3-α. In one embodiment, a bacterial strain of the species Megasphaeramassiliensis is for use in treating or preventing diseases by increasingthe expression and/or activity of MIP3-α. In one embodiment, a bacterialstrain of the species Megasphaera massiliensis is for use in promotingthe immune response by increasing the expression and/or activity ofMIP3-α.

The Examples show that administration of the compositions of theinvention can lead to an increase in expression of Tumour NecrosisFactor alpha (TNF-α). TNF-α is a pro-inflammatory cytokine which isknown to be involved in various signalling pathways to promote celldeath. TNF-α initiates apoptosis by binding to its cognate receptor,TNFR-1, which leads to a cascade of cleavage events in the apoptoticpathway [28]. TNF-α can also trigger necrosis via a RIP kinase-dependentmechanism [29]. Since administration of the compositions of theinvention show an increase in TNF-α expression, compositions of theinvention may be useful in the treatment of diseases, in particular foruse in treating or preventing diseases characterised by a decrease inexpression of by TNF-α. In one embodiment, the compositions of theinvention are for use in treating diseases characterised by decreasedTNF-α expression. In a particular embodiment, the compositions of theinvention are for use in treating or preventing diseases characterisedby a decrease in the expression and/or activity of TNF-α. In oneembodiment, the compositions of the invention may be useful for treatingor preventing diseases by increasing the expression and/or activity ofTNF-α. In one embodiment, compositions of the invention are for use inpromoting the immune response by increasing the expression and/oractivity of TNF-α. In certain embodiments, a bacterial strain of thespecies Megasphaera massiliensis is for use in the treatment of diseasescharacterised by a decrease in expression of and/or activity of TNF-α.In one embodiment, a bacterial strain of the species Megasphaeramassiliensis is for use in treating or preventing diseases by increasingthe expression and/or activity of TNF-α. In one embodiment, a bacterialstrain of the species Megasphaera massiliensis is for use in promotingthe immune response by increasing the expression and/or activity ofTNF-α.

The Examples also show that administration of the compositions of theinvention can lead to an increase in expression of IL-6 in PBMCs. IL-6 apro-inflammatory cytokine that is produced during inflammation, andpromotes the differentiation of immature CD4+ T cells and thedifferentiation of CD8+ T cells into cytotoxic T cells [30]. Sinceadministration of the compositions of the invention were shown toincrease the expression of IL-6, compositions of the invention may beuseful in the treatment of diseases characterised by a decrease inexpression of IL-6. In a particular embodiment, the compositions of theinvention are for use in treating or preventing diseases characterisedby a decrease in the expression and/or activity of IL-6. In oneembodiment, the compositions of the invention are for use in treating orpreventing diseases by increasing the expression and/or activity ofIL-6. In one embodiment, compositions of the invention are for use inpromoting the immune response by increasing the expression and/oractivity of IL-6. In certain embodiments, a bacterial strain of thespecies Megasphaera massiliensis is for use in the treatment of diseasescharacterised by a decrease in expression of and/or activity of IL-6. Inone embodiment, a bacterial strain of the species Megasphaeramassiliensis is for use in treating or preventing diseases by increasingthe expression and/or activity of IL-6. In one embodiment, a bacterialstrain of the species Megasphaera massiliensis is for use in promotingthe immune response by increasing the expression and/or activity ofIL-6.

Bettelli et al. [31] reported that IL-6 inhibits the production ofTregs. Since the Examples show that compositions of the inventionincrease the expression of IL-6, compositions of the invention mayselectively decrease the number or percentage of Tregs by increasing theexpression of IL-6. In one embodiment, compositions of the invention arefor use in immunostimulation by increasing the expression of IL-6. Inanother embodiment, compositions of the invention are for use inimmunostimulation by decreasing the number or percentage of Tregs. Inone embodiment, a bacterial strain of the species Megasphaeramassiliensis is for use in immunostimulation by increasing theexpression of IL-6. In another embodiment, a bacterial strain of thespecies Megasphaera massiliensis is for use in immunostimulation bydecreasing the number or percentage of Tregs.

The Examples also show that administration of the compositions of theinvention can lead to an increase in expression of IL-8 (see Example 8).IL-8 is a pro-inflammatory cytokine secreted predominantly bymacrophages with immune-stimulatory effects. It induces chemotaxis intarget cells, primarily neutrophils but also other granulocytes, causingthem to migrate toward the site of infection. IL-8 also stimulatesphagocytosis. Since administration of the compositions of the inventionwere shown to increase the expression of IL-8, compositions of theinvention may be useful in the treatment of diseases characterised by adecrease in expression of IL-8. In a particular embodiment, thecompositions of the invention are for use in treating or preventingdiseases characterised by a decrease in the expression and/or activityof IL-8. In one embodiment, the compositions of the invention are foruse in treating or preventing diseases by increasing the expressionand/or activity of IL-8. In one embodiment, compositions of theinvention are for use in promoting the immune response by increasing theexpression and/or activity of IL-8. In certain embodiments, a bacterialstrain of the species Megasphaera massiliensis is for use in thetreatment of diseases characterised by a decrease in expression of IL-8and/or activity of IL-8. In one embodiment, a bacterial strain of thespecies Megasphaera massiliensis is for use in treating or preventingdiseases by increasing the expression and/or activity of IL-8. In oneembodiment, a bacterial strain of the species Megasphaera massiliensisis for use in promoting the immune response by increasing the expressionand/or activity of IL-8.

The Examples also show that administration of the compositions of theinvention can lead to an increase in expression of CD11b (see Example12). CD11b is a pro-inflammatory cytokine with immune-stimulatoryeffects. CD11b is expressed on the surface of many leukocytes involvedin the innate immune system and mediates inflammation by regulatingleukocyte adhesion and migration. CD11b has been implicated in severalimmune processes, for example phagocytosis, cell-mediated cytotoxicity,chemotaxis and cellular activation. Since administration of thecompositions of the invention were shown to increase the expression ofCD11b, compositions of the invention may be useful in the treatment ofdiseases characterised by a decrease in expression of CD11b. In aparticular embodiment, the compositions of the invention are for use intreating or preventing diseases characterised by a decrease in theexpression and/or activity of CD11b. In one embodiment, the compositionsof the invention are for use in treating or preventing diseases byincreasing the expression and/or activity of CD11b. In one embodiment,compositions of the invention are for use in promoting the immuneresponse by increasing the expression and/or activity of CD11b. Incertain embodiments, a bacterial strain of the species Megasphaeramassiliensis is for use in the treatment of diseases characterised by adecrease in expression of CD11b and/or activity of CD11b. In oneembodiment, a bacterial strain of the species Megasphaera massiliensisis for use in treating or preventing diseases by increasing theexpression and/or activity of CD11b. In one embodiment, a bacterialstrain of the species Megasphaera massiliensis is for use in promotingthe immune response by increasing the expression and/or activity ofCD11b.

The Examples show that compositions of the invention can induceNF-κB-Ap1 promoter activation (see FIG. 61 ). NF-κB is involved in theactivation of the immune response in particular by stimulating theexpression of mediators of inflammation and cytokines involved in theimmune response, for example IL-6. As outlined above, an increase in theexpression of IL-6 helps to stimulate the immune system and thus theactivation of the NF-κB pathway has immunostimulatory activity.Accordingly, in certain embodiments, the compositions of the inventionactivate NF-κB signalling and thus stimulate the immune system. Incertain embodiments, the compositions of the invention stimulateexpression of mediators of inflammation and immunostimulatory cytokinesby increasing the activation of the NF-κB promoter.

Cancer

In preferred embodiments, the compositions of the invention are for usein treating or preventing cancer. In a particular embodiment,compositions of the invention are for use in treating or preventingbrain cancer, in particular neuroblastoma. In a particular embodiment,compositions of the invention are for use in treating or preventingmelanoma, in particular metastatic melanoma. In certain embodiments, thecompositions of the invention are for use in treating or preventingbrain cancer. In certain embodiments, the compositions of the inventionare for use in treating or preventing neuroblastoma. In certainembodiments, the compositions of the invention are for use in treatingor preventing melanoma. In certain embodiments, the compositions of theinvention are for use in treating or preventing metastatic melanoma. Ina most preferred embodiment, the composition of the invention comprisesa bacterial strain of the species Megasphaera massiliensis and is foruse in treating or preventing brain cancer, in particular neuroblastoma.In a further most preferred embodiment, the composition of the inventioncomprises a bacterial strain of the species Megasphaera massiliensis andis for use in treating or preventing melanoma, in particular metastaticmelanoma.

The Examples (Example 1) demonstrate that administration of thecompositions of the invention can lead to an increase in Class III betatubulin (β3 Tubulin) expression in undifferentiated neuroblastoma cells.β3 tubulin is widely known as a neuronal marker [32]. The Examples alsodemonstrate that administration of the compositions of the invention canlead to an increase in Microtubule-associated protein 2 (MAP2)expression in undifferentiated neuroblastoma cells. MAP2 ispredominantly expressed in neurons and functions to stabilisemicrotubules, to promote the development of dendrites and for neuriteoutgrowth [33]. MAP2 is known as a marker of differentiated neurons.

Agents which cause cell differentiation have been associated with cancertherapeutics, since administration of cell-differentiating agents hasbeen correlated with the inhibition of tumour growth [34]. Therefore,the compositions of the invention may be useful in treating cancer. In aparticular embodiment, compositions of the invention are for use intreating cancers by inducing cell differentiation, in particularneuronal differentiation. In one embodiment, compositions of theinvention are for use in treating brain cancer by inducing neuronaldifferentiation, in particular the treatment of neuroblastoma.

Furthermore, MAP2 has been found to be highly expressed in primarycutaneous melanomas, but has reduced expression in metastatic melanomas[35]. It has been proposed that increased expression ofmicrotubule-stabilizing proteins or treatment with microtubulestabilizing proteins such as MAP2 may interfere the dynamic instabilityof microtubules which is required during cell division. Therefore,upregulation of MAP2 is thought to hamper cell division and delay tumourgrowth in cancer [35]. Therefore, compositions of the invention may beuseful for treating cancer, in particular metastatic cancers. In oneembodiment, the compositions of the invention are for use in a method oftreating cancer. In certain embodiments, the compositions of theinvention are for use in treating or preventing cancers mediated bydecreased MAP2 expression. In certain embodiments, the compositions ofthe invention are for use in treating or preventing cancerscharacterised by decreased or absent MAP2 expression. In certainembodiments, the compositions of the invention are for use in increasingMAP2 expression in the treatment of cancer. In a preferred embodiment,the compositions of the invention are for use in treating or preventingmelanoma. In a particular embodiment, the compositions of the inventionare for use in treating or preventing metastatic melanoma.

In certain embodiments, the therapeutic combinations of the inventionare for use in treating or preventing melanoma. According to someembodiments, the therapeutic combinations of the invention have aneffect on melanocytes and may be effective for treating melanoma. Incertain embodiments, the therapeutic combinations of the invention arefor use in reducing tumour size, reducing tumour growth, or reducingangiogenesis in the treatment of melanoma.

In particular, the Examples show that administration of the compositionsof the invention can lead to an increase in MAP2 expression inundifferentiated neuroblastoma cells. Since MAP2 is widely known as amarker of differentiated neurons and its expression has been shown tohave implications in cancer, the compositions of the invention may beparticularly useful for treating brain cancer, such as neuroblastoma. Inone embodiment, the compositions of the invention are for use in amethod of treating brain cancer. In a preferred embodiment, thecompositions of the invention are for use in a method of treatingneuroblastoma.

Furthermore, the Examples also show that administration of thecompositions of the invention can lead to a significant decrease inDopamine Receptor D2 (DRD2) expression (see Example 2 and FIG. 3 ). DRD2is a G-protein coupled receptor (GPCR) and it is part of the dopaminereceptor family. DRD2 is involved in signalling pathways which promotecell survival, and is therefore associated with cancer. Theoverexpression or upregulation of DRD2 has been implicated in severaltypes of cancer, as malignant cells show an increased expression of DRD2when compared to normal cells [36]. It has been shown that inhibitingDRD2 through DRD2-specific antagonists has anti-tumour effects. DRD2antagonists have been shown to have anti-tumour efficacy in manycancers, including breast cancer [37][38], glioblastoma [39][40][41],neuroblastoma [42], hepatocellular carcinoma [43], lung cancer, prostatecancer [44], cervical cancer [45], ovarian cancer [46], lymphoma [47]and gastric cancer [48]. Therefore, compositions that decrease theexpression level of DRD2 may be useful for the treatment of cancer.Since administration of the compositions of the invention were shown todecrease DRD2 expression, compositions of the invention may be useful inthe treatment of cancer, in particular for use in treating or preventingcancers characterised by increased DRD2 expression. In one embodiment,the compositions of the invention may be useful for treating orpreventing cancers characterised by increased expression and/or activityof DRD2. In certain embodiments, the compositions of the invention arefor use in decreasing DRD2 expression and/or activity in the treatmentof cancer. In one embodiment, the compositions of the invention may beuseful for treating cancer, in particular breast cancer, ovarian cancer,cervical cancer, brain cancer, in particular glioblastoma andneuroblastoma, carcinoma, in particular hepatocellular carcinoma, lungcancer, prostate cancer lymphoma and/or gastric cancer. In oneembodiment, the compositions of the invention may be useful for treatingor preventing cancer by decreasing the level and/or activity of DRD2.

Prabhu et al. reported that ONC201, an antagonist of DRD2, has shownefficacy in shrinking tumours in glioblastoma models. DRD2 expression isupregulated in glioblastoma tumours, and therefore DRD2 is an attractivetarget for cancer therapeutics [49]. In certain embodiments, thecompositions of the invention are for use in treating or preventingglioblastoma.

The Examples also show that administration of the compositions of theinvention can lead to an increase in Caspase 3 (Casp3) expression inSH-SY5Y cells. Caspases are part of the cysteine protease family and areknown to promote apoptosis. Casp3 is known as an “executioner caspase”,which plays an important role in the cleavage cascade of cellularproteins in the apoptotic pathway. Downregulation of Casp3 expressionhas previously been shown in cancers from breast, ovarian and cervicaltumour tissues, and it is thought that decreased expression of Casp3promotes cell survival in cancerous tissue [50]. Therefore, compositionsthat increase the expression level of executioner caspases, inparticular Casp3, may be useful for the treatment of cancer. Sinceadministration of the compositions of the invention were shown toincrease Casp3 expression, compositions of the invention may be usefulin the treatment of cancer, in particular for use in treating orpreventing cancers mediated by Casp3 expression. In certain embodiments,the compositions of the invention are for use in treating or preventingcancers characterised by decreased or absent Casp3 expression. In oneembodiment, the compositions of the invention may be useful for treatingcancers characterised by decreased or absent executioner caspaseexpression. In a particular embodiment, the compositions of theinvention may be useful for treating or preventing cancers characterisedby decreased or absent expression of Casp3. In certain embodiments, thecompositions of the invention are for use in increasing Casp3 expressionin the treatment of cancer. In one embodiment, the compositions of theinvention may be useful for treating cancer, in particular breastcancer, ovarian cancer and/or cervical cancer. In one embodiment, thecompositions of the invention may be useful for treating cancer byincreasing the level and/or activity of Casp3.

Furthermore, caspases have been reported to be involved in processesother than apoptosis, such as cell differentiation [51]. The Examples(Example 1 and Example 3) demonstrate that administration of thecompositions of the invention can lead to an increase in the expressionof neuronal markers β3 Tubulin and MAP2, and also increase theexpression of Casp3 in undifferentiated neuroblastoma cells. Sincecompositions of the invention can lead to an increase in expression ofneuronal markers and proteins known to play a role in celldifferentiation, compositions of the invention may be useful in thedifferentiation of neurons from undifferentiated cells.

The Examples also show that administration of the compositions of theinvention can lead to a decrease in cell viability in undifferentiatedneuroblastoma cells (FIG. 5 ). In particular, the Examples show thatadministration of MRx0029 at a concentration of 5% or 10% causes asignificant, dose-dependent decrease in cell viability (FIG. 5 ).

It is known that a decrease in cell viability, or increased cell deathof cancerous cells is a target for cancer treatment [52]. Therefore,compositions that decrease cell viability in cancer cell lines, such asneuroblastoma cell lines, may be useful for the treatment of cancer. Inone embodiment, the compositions of the invention are for use in thetreatment of cancer by decreasing cell viability. In another embodiment,compositions of the invention are for use in the treatment of cancer byincreasing cell death.

Furthermore, since the Examples show that compositions of the inventionboth increase Casp3 expression and decrease cell viability (FIG. 4 andFIG. 5 ), it is proposed that the compositions of the invention decreasecell viability by upregulating apoptosis. In one embodiment,compositions of the invention are for use in upregulating apoptosis. Inanother embodiment, compositions of the invention are for use in thetreatment of cancer by increasing cell death, in particular byincreasing apoptosis. In one embodiment, compositions of the inventionare for use in treating cancers by decreasing cell viability. In oneembodiment, compositions of the invention are for use in treatingcancers by decreasing cell viability. In one embodiment, compositions ofthe invention are for use in treating cancers by upregulating apoptosis.In a particular embodiment, compositions of the invention are for use intreating cancers by upregulating apoptosis. In a particular embodiment,compositions of the invention are for use in treating cancerscharacterised by decreased or absent Casp3 expression by upregulatingapoptosis. In certain embodiments, the compositions of the invention arefor use in increasing apoptosis in the treatment of cancer. In certainembodiments, the compositions of the invention are for use in decreasingcell viability in the treatment of cancer.

The Examples show that compositions of the invention increase both Casp3and MAP2 expression. Therefore, Casp3 upregulation and MAP2 upregulationmay be related.

Histone acetylation and deacetylation are important epigeneticregulators of gene expression. Epigenetic regulation is a powerful toolfor regulating all aspects of cell function. Histone deacetylases (HDAC)repress gene expression by removing acetyl groups from an ε-N-acetyllysine amino acid on a histone, allowing the histones to wrap the DNAmore tightly and resulting in transcriptional suppression throughnucleosomal inaccessibility. HDAC has 18 isoforms which are organisedinto four classes: Class I, II, III and IV. Alterations in HDAC levelshave been observed in many disease types including for example cancers,infectious diseases, inflammatory diseases and neurodegenerativediseases [53, 54, 55].

HDAC inhibitors (HDACi) are an emerging class of promising anti-cancerdrugs that have been shown to cause growth arrest, differentiation,apoptosis, reduction of angiogenesis and modulation of the immuneresponse in a variety of cancer cell lines [56, 57, 58, 59]. Althoughthe precise mechanism by which the clinical activity of these agents ismediated remains unclear, a wide range of HDACi are currently beinginvestigated as potential anticancer agents. Furthermore, due to thedemonstrable anticancer activity in both in vitro and in vivo studies,many HDACi have progressed rapidly through clinical development, eitheras monotherapies or in combination with other anticancer agents [60].Among them, vorinostat (Zolinza™), romidepsin (Istodax™), and belinostat(Beleodaq™) have received approval from the US FDA for the treatment oflymphoma. Lymphoma and other blood cancers (also called haematologiccancers or haematologic malignancies) are particularly sensitive toHDACi. The gut microbiota, with its immense diversity and metaboliccapacity, represents a huge metabolic reservoir for production of a vastvariety of molecules with potential effects on HDAC activity. Fewstudies have assessed the HDAC inhibitory activity ofmicrobially-derived metabolites other than butyrate, which has beenshown to inhibit HDAC and is associated with improvement of motorfunction in Huntington's disease [61].

The Examples show that compositions of the invention inhibit HDACactivity, in particular Class I HDAC, for example HDAC2 (Examples 9 and10). Accordingly, in certain embodiments, the compositions of theinvention modulate HDAC activity. In certain embodiments, thecompositions of the invention reduce HDAC activity. In certainembodiments, the compositions of the invention inhibit HDAC. In certainembodiments, the compositions of the invention are HDACi. In preferredembodiments, the compositions of the invention reduce Class I HDACactivity. In certain embodiments, the compositions of the inventionreduce Class II HDAC activity. In certain embodiments, the compositionsof the invention reduce Class III HDAC activity. In certain embodiments,the compositions of the invention reduce Class IV HDAC activity. Incertain embodiments, the compositions of the invention reduce HDAC1activity. In preferred embodiments, the compositions of the inventionreduce HDAC2 activity. In certain embodiments, the compositions of theinvention reduce HDAC3 activity. In certain embodiments, thecompositions of the invention reduce HDAC activity through production ofvaleric acid. In certain embodiments, the compositions of the inventionreduce HDAC activity through production of sodium butyrate. In preferredembodiments, the compositions of the invention are for use in treatingor preventing cancer by reducing HDAC activity. In preferredembodiments, the compositions of the invention are for use in treatingor preventing HDAC associated cancers. In certain embodiments, thecompositions of the invention are for use in treating or preventingmetastatic melanoma, breast cancer, ovarian cancer, cervical cancer,neuroblastoma, glioblastoma, carcinoma, lung cancer, chronic lymphocyteleukemia, prostate cancer, lymphoma, colorectal cancer, haematologicalmalignancies and/or gastric cancer by reducing HDAC activity. Inpreferred embodiments, the compositions of the invention are for use intreating or preventing haematological malignancies by reducing HDACactivity.

In certain embodiments, the HDAC inhibitory activity of the compositionsof the invention results in growth arrest. In certain embodiments, theHDAC inhibitory activity of the compositions of the invention results incell-cycle arrest. In certain embodiments, the HDAC inhibitory activityof the compositions of the invention results in cell differentiation. Incertain embodiments, the HDAC inhibitory activity of the compositions ofthe invention results in apoptosis. In certain embodiments, the HDACinhibitory activity of the compositions of the invention results inreduction of angiogenesis. In certain embodiments, the HDAC inhibitoryactivity of the compositions of the invention results in modulation ofthe immune response. In certain embodiments, the compositions of theinvention are for use in reducing HDAC activity as a monotherapy. Incertain embodiments, the compositions of the invention are for use inreducing HDAC activity as a combination therapy. In certain embodiments,the compositions of the invention are for use in combination withanother anticancer agent. In certain embodiments, the compositions ofthe invention are for use in combination with more than one otheranticancer agent. In certain embodiments, the compositions of theinvention are for use in combination with a chemotherapeutic agent. Incertain embodiments, the compositions of the invention are for use incombination with a proteasome inhibitor. In further aspects, thecompositions of the invention are epigenetic regulators. In certainembodiments, the compositions of the invention are for use in treatingor preventing diseases characterised by epigenetic aberrations.

In certain embodiments, a bacterial strain of the species Megasphaeramassiliensis modulates HDAC activity. In certain embodiments, abacterial strain of the species Megasphaera massiliensis reduces HDACactivity. In certain embodiments, a bacterial strain of the speciesMegasphaera massiliensis inhibits HDAC. In certain embodiments, abacterial strain of the species Megasphaera massiliensis is a HDACi. Inpreferred embodiments, a bacterial strain of the species Megasphaeramassiliensis reduces Class I HDAC activity. In certain embodiments, abacterial strain of the species Megasphaera massiliensis reduces ClassII HDAC activity. In certain embodiments, a bacterial strain of thespecies Megasphaera massiliensis reduces Class III HDAC activity. Incertain embodiments, a bacterial strain of the species Megasphaeramassiliensis reduces Class IV HDAC activity. In certain embodiments, abacterial strain of the species Megasphaera massiliensis reduces HDAC1activity. In preferred embodiments, a bacterial strain of the speciesMegasphaera massiliensis reduces HDAC2 activity. In certain embodiments,a bacterial strain of the species Megasphaera massiliensis reduces HDAC3activity. In certain embodiments, a bacterial strain of the speciesMegasphaera massiliensis reduces HDAC activity through production ofvaleric acid. In certain embodiments, a bacterial strain of the speciesMegasphaera massiliensis reduces HDAC activity through production ofsodium butyrate. In preferred embodiments, a bacterial strain of thespecies Megasphaera massiliensis is for use in treating or preventingcancer by reducing HDAC activity. In preferred embodiments, a bacterialstrain of the species Megasphaera massiliensis is for use in treating orpreventing HDAC associated cancers. In certain embodiments, a bacterialstrain of the species Megasphaera massiliensis is for use in treating orpreventing metastatic melanoma, breast cancer, ovarian cancer, cervicalcancer, neuroblastoma, glioblastoma, carcinoma, lung cancer, chroniclymphocyte leukemia, prostate cancer, lymphoma, colorectal cancer,haematological malignancies and/or gastric cancer by reducing HDACactivity. In preferred embodiments, a bacterial strain of the speciesMegasphaera massiliensis is for use in treating or preventinghaematological malignancies by reducing HDAC activity.

In certain embodiments, the HDAC inhibitory activity of the bacterialstrain of the species Megasphaera massiliensis results in growth arrest.In certain embodiments, the HDAC inhibitory activity of the bacterialstrain of the species Megasphaera massiliensis results in cell-cyclearrest. In certain embodiments, the HDAC inhibitory activity of thebacterial strain of the species Megasphaera massiliensis results in celldifferentiation. In certain embodiments, the HDAC inhibitory activity ofthe bacterial strain of the species Megasphaera massiliensis results inapoptosis. In certain embodiments, a bacterial strain of the speciesMegasphaera massiliensis results in reduction of angiogenesis. Incertain embodiments, the HDAC inhibitory activity of a bacterial strainof the species Megasphaera massiliensis results in modulation of theimmune response. In certain embodiments, a bacterial strain of thespecies Megasphaera massiliensis is for use in reducing HDAC activity asa monotherapy. In certain embodiments, a bacterial strain of the speciesMegasphaera massiliensis is for use in reducing HDAC activity as acombination therapy. In certain embodiments, a bacterial strain of thespecies Megasphaera massiliensis is for use in combination with anotheranticancer agent. In certain embodiments, a bacterial strain of thespecies Megasphaera massiliensis is for use in combination with morethan one other anticancer agent. In certain embodiments, a bacterialstrain of the species Megasphaera massiliensis is for use in combinationwith a chemotherapeutic agent. In certain embodiments, a bacterialstrain of the species Megasphaera massiliensis is for use in combinationwith a proteasome inhibitor. In further aspects, a bacterial strain ofthe species Megasphaera massiliensis is an epigenetic regulator. Incertain embodiments, a bacterial strain of the species Megasphaeramassiliensis is for use in treating or preventing diseases characterisedby epigenetic aberrations.

Compositions of the invention are able to regulate epithelialpermeability by modifying intracellular signal transduction involved inthe expression and localization of proteins involved in gut barrierfunction. In particular, compositions of the invention enhance Occludin,Villin, Tight Junction Protein 1 (TJP1) and Tight Junction Protein 2(TJP2) mRNA expression. Compositions of the invention therefore functionto increase gut barrier function and reduce gut permeability (Example11). In certain embodiments, the compositions of the invention are foruse in increasing gut barrier function. In certain embodiments, thecompositions of the invention are for use in reducing gut permeability.In certain embodiments, the compositions of the invention are for use intreating or preventing reduction of gut barrier function. In certainembodiments, the compositions of the invention are for use in treatingor preventing increased gut permeability. In certain embodiments, thecompositions of the invention are for use in treating or preventingdiseases or conditions that are characterised by reduction of gutbarrier function. In certain embodiments, the compositions of theinvention are for use in treating or preventing diseases or conditionsthat are characterised by increased gut permeability. In certainembodiments, the compositions of the invention are for use in treatingor preventing reduction of gut barrier function resulting fromradiotherapy or chemotherapy. In certain embodiments, the compositionsof the invention are for use in treating or preventing increased gutpermeability resulting from radiotherapy or chemotherapy. In certainembodiments, the compositions of the invention are for use in treatingor preventing cachexia by increasing gut barrier function. In certainembodiments, the compositions of the invention are for use in treatingor preventing cachexia by reducing gut permeability. In certainembodiments the cachexia is cancer cachexia.

In certain embodiments, a bacterial strain of the species Megasphaeramassiliensis is for use in increasing gut barrier function. In certainembodiments, a bacterial strain of the species Megasphaera massiliensisis for use in reducing gut permeability. In certain embodiments, abacterial strain of the species Megasphaera massiliensis is for use intreating or preventing reduction of gut barrier function. In certainembodiments, a bacterial strain of the species Megasphaera massiliensisis for use in treating or preventing increased gut permeability. Incertain embodiments, a bacterial strain of the species Megasphaeramassiliensis is for use in treating or preventing diseases or conditionsthat are characterised by reduction of gut barrier function. In certainembodiments, a bacterial strain of the species Megasphaera massiliensisis for use in treating or preventing diseases or conditions that arecharacterised by increased gut permeability. In certain embodiments, abacterial strain of the species Megasphaera massiliensis is for use intreating or preventing reduction of gut barrier function resulting fromradiotherapy or chemotherapy. In certain embodiments, a bacterial strainof the species Megasphaera massiliensis is for use in treating orpreventing increased gut permeability resulting from radiotherapy orchemotherapy. In certain embodiments, a bacterial strain of the speciesMegasphaera massiliensis is for use in treating or preventing cachexiaby increasing gut barrier function. In certain embodiments a bacterialstrain of the species Megasphaera massiliensis is for use in treating orpreventing cachexia by reducing gut permeability. In certain embodimentsthe cachexia is cancer cachexia.

In preferred embodiments, the compositions are for treating cancer in apatient undergoing radiotherapy or chemotherapy. In such embodiments,the composition may be administered prior to, during, or afterradiotherapy or chemotherapy. Patients undergoing radiotherapy orchemotherapy should not be administered any agent which induces a leakygut, but Megasphaera massiliensis promotes gut-barrier function [62], sothe compositions of the invention are particularly suitable for treatingpatients undergoing radiotherapy or chemotherapy. Activation of TLR-5has been shown to ameliorate radiation-induced epithelial damage in vivo[63]. The compositions of the invention also activate the immune system.In some embodiments, the compositions of the invention are for use intreating radiotherapy-induced damage. In certain embodiments, abacterial strain of the species Megasphaera massiliensis is for use intreating radiotherapy-induced damage.

In some embodiments, compositions of the invention can lead to areduction in tumour growth.

In certain embodiments, treatment with the compositions of the inventionresults in a reduction in tumour size or a reduction in tumour growth.In certain embodiments, the compositions of the invention are for use inreducing tumour size or reducing tumour growth. The compositions of theinvention may be effective for reducing tumour size or growth. Incertain embodiments, the compositions of the invention are for use inpatients with solid tumours. In certain embodiments, the compositions ofthe invention are for use in reducing or preventing angiogenesis in thetreatment of cancer. The compositions of the invention may have aneffect on the immune or inflammatory systems, which have central rolesin angiogenesis. The compositions of the invention may haveanti-metastatic activity. A bacterial strain of the species Megasphaeramassiliensis is may have anti-metastatic activity. In certainembodiments, the compositions of the invention are for use in preventingmetastasis. In certain embodiments, a bacterial strain of the speciesMegasphaera massiliensis is for use in preventing metastasis.

The Examples show that administration of the compositions of theinvention can lead to a decrease in the percentage of Tregs in PBMCs(FIG. 6C). Tregs have been implicated in cancer, and infiltration ofTregs in tumour tissue has been linked to poor prognosis [64]. Sinceadministration of the compositions of the invention were shown toselectively reduce the population of Tregs (FIG. 6C), compositions ofthe invention may be useful in the treatment of cancer. In oneembodiment, the compositions of the invention are for use in treating orpreventing cancers characterised by an increase in the percentage ofTregs in a cell population. In one embodiment, the compositions of theinvention may be useful for treating or preventing cancers characterisedby an increase in the percentage of CD4+CD25+CD127− cells in a cellpopulation. In one embodiment, the compositions of the invention are foruse in treating or preventing cancers by decreasing the number orpercentage of Tregs, in particular in cancerous tissue. In oneembodiment, compositions of the invention are for use in treating cancerby the selective reduction of Tregs.

It has been proposed that selectively reducing the number of Tregs andactivating effector T cells, such as CD8+ T cells, will be an effectivecancer therapy [64]. The Examples also show that administration of thecompositions of the invention can lead to an increase in the ratio ofCD8 cells to Treg cells. Since administration of the compositions of theinvention were shown to increase the ratio of both CD8/Treg cells andactivated CD8/Treg cells (FIG. 6G and FIG. 6H), compositions of theinvention may be useful in the treatment of cancer. In one embodiment,the compositions of the invention are for use in treating or preventingcancers characterised by a decrease in the ratio of CD8/Treg and/oractivated CD8/Treg cells. In one embodiment, the compositions of theinvention are for use in treating or preventing cancers characterised bya decrease in the ratio of CD8/Treg cells. In one embodiment, thecompositions of the invention are for use in treating or preventingcancers characterised by a decrease in the ratio of activated CD8/Tregcells. In one embodiment, the compositions of the invention are for usein treating or preventing cancer by decreasing the percentage of Tregsin cell populations, thereby increasing the ratio of CD8/Treg cells. Inanother embodiment, the compositions of the invention are for use intreating or preventing cancer by decreasing the percentage of Tregs incell populations, thereby increasing the ratio of activated CD8/Tregcells. In one embodiment, compositions of the invention are for use intreating cancer by increasing the ratio of CD8/Treg cells. In oneembodiment, compositions of the invention are for use in treating cancerby increasing the ratio of activated CD8/Treg cells.

The Examples also show that administration of the compositions of theinvention can lead to an increase in expression of IL-1β. In inColitis-associated cancer, decreased expression of IL-1β at tumour siteshas been linked to symptoms such as increased disease outcome andmorbidity [65]. Since administration of the compositions of theinvention were shown to increase the expression of IL-1β, compositionsof the invention may be useful in the treatment of cancer. In oneembodiment, the compositions of the invention are for use in treating orpreventing cancers characterised by decreased or absent expression ofIL-1β. In one embodiment, the compositions of the invention are for usein treating or preventing cancer by increasing the expression of IL-1β.

The Examples show that administration of the compositions of theinvention can lead to an increase in expression of Tumour NecrosisFactor alpha (TNF-α). TNF-α is a pro-inflammatory cytokine which isknown to be involved in various signalling pathways to promote celldeath. TNF-α initiates apoptosis by binding to its cognate receptor,TNFR-1, which leads to a cascade of cleavage events in the apoptoticpathway. TNF-α can also trigger necrosis via a RIP kinase-dependentmechanism. Since many types of cancers have defective apoptotic andnecrotic pathways, and TNF-α is known to a mediate these cell deathpathways, TNF-α is a potential target for cancer therapy. Sinceadministration of the compositions of the invention were shown toincrease TNF-α expression, compositions of the invention may be usefulin the treatment of cancer, in particular for use in treating orpreventing cancers mediated by TNF-α expression. In one embodiment, thecompositions of the invention may be useful for treating cancersmediated by TNF-α expression, in particular cancers with decreasedexpression and/or activity of TNF-α. In one embodiment, the compositionsof the invention may be useful for treating cancer. In one embodiment,the compositions of the invention may be useful for treating cancer byincreasing the level and/or activity of TNF-α.

In preferred embodiments, the compositions of the invention are for usein treating or preventing breast cancer. In certain embodiments, thecompositions of the invention are for use in reducing tumour size,reducing tumour growth, or reducing angiogenesis in the treatment ofbreast cancer. In preferred embodiments, a bacterial strain of thespecies Megasphaera massiliensis is for use in treating or preventingbreast cancer. In certain embodiments, a bacterial strain of the speciesMegasphaera massiliensis is for use in reducing tumour size, reducingtumour growth, or reducing angiogenesis in the treatment of breastcancer. In preferred embodiments the cancer is mammary carcinoma. Inpreferred embodiments the cancer is stage IV breast cancer.

In certain embodiments, the compositions of the invention are for use intreating or preventing lung cancer. In certain embodiments, thecompositions of the invention are for use in reducing tumour size,reducing tumour growth, or reducing angiogenesis in the treatment oflung cancer. In certain embodiments, a bacterial strain of the speciesMegasphaera massiliensis is for use in treating or preventing lungcancer. In certain embodiments, a bacterial strain of the speciesMegasphaera massiliensis is for use in reducing tumour size, reducingtumour growth, or reducing angiogenesis in the treatment of lung cancer.In preferred embodiments the cancer is lung carcinoma.

In certain embodiments, the compositions of the invention are for use intreating or preventing liver cancer. In certain embodiments, thecompositions of the invention are for use in reducing tumour size,reducing tumour growth, or reducing angiogenesis in the treatment ofliver cancer. In certain embodiments, a bacterial strain of the speciesMegasphaera massiliensis is for use in treating or preventing livercancer. In certain embodiments, a bacterial strain of the speciesMegasphaera massiliensis is for use in reducing tumour size, reducingtumour growth, or reducing angiogenesis in the treatment of livercancer. In preferred embodiments the cancer is hepatoma (hepatocellularcarcinoma).

In preferred embodiments, the compositions of the invention are for usein treating or preventing melanoma. In certain embodiments, thecompositions of the invention are for use in reducing tumour size,reducing tumour growth, or reducing angiogenesis in the treatment ofmelanoma. In preferred embodiments, a bacterial strain of the speciesMegasphaera massiliensis is for use in treating or preventing melanoma.In certain embodiments, a bacterial strain of the species Megasphaeramassiliensis is for use in reducing tumour size, reducing tumour growth,or reducing angiogenesis in the treatment of melanoma. In preferredembodiments the melanoma is metastatic melanoma.

In preferred embodiments, the compositions of the invention are for usein treating or preventing ovarian cancer. In certain embodiments, thecompositions of the invention are for use in reducing tumour size,reducing tumour growth, or reducing angiogenesis in the treatment ofovarian cancer. In preferred embodiments, a bacterial strain of thespecies Megasphaera massiliensis is for use in treating or preventingovarian cancer. In certain embodiments, a bacterial strain of thespecies Megasphaera massiliensis is for use in reducing tumour size,reducing tumour growth, or reducing angiogenesis in the treatment ofovarian cancer.

In preferred embodiments, the compositions of the invention are for usein treating or preventing cervical cancer. In certain embodiments, thecompositions of the invention are for use in reducing tumour size,reducing tumour growth, or reducing angiogenesis in the treatment ofcervical cancer. In preferred embodiments, a bacterial strain of thespecies Megasphaera massiliensis is for use in treating or preventingcervical cancer. In certain embodiments, a bacterial strain of thespecies Megasphaera massiliensis is for use in reducing tumour size,reducing tumour growth, or reducing angiogenesis in the treatment ofcervical cancer.

In preferred embodiments, the compositions of the invention are for usein treating or preventing neuroblastoma. In certain embodiments, thecompositions of the invention are for use in reducing tumour size,reducing tumour growth, or reducing angiogenesis in the treatment ofneuroblastoma. In preferred embodiments, a bacterial strain of thespecies Megasphaera massiliensis is for use in treating or preventingneuroblastoma. In certain embodiments, a bacterial strain of the speciesMegasphaera massiliensis is for use in reducing tumour size, reducingtumour growth, or reducing angiogenesis in the treatment ofneuroblastoma.

In preferred embodiments, the compositions of the invention are for usein treating or preventing glioblastoma. In certain embodiments, thecompositions of the invention are for use in reducing tumour size,reducing tumour growth, or reducing angiogenesis in the treatment ofglioblastoma. In preferred embodiments, a bacterial strain of thespecies Megasphaera massiliensis is for use in treating or preventingglioblastoma. In certain embodiments, a bacterial strain of the speciesMegasphaera massiliensis is for use in reducing tumour size, reducingtumour growth, or reducing angiogenesis in the treatment ofglioblastoma.

In preferred embodiments, the compositions of the invention are for usein treating or preventing prostate cancer. In certain embodiments, thecompositions of the invention are for use in reducing tumour size,reducing tumour growth, or reducing angiogenesis in the treatment ofprostate cancer. In preferred embodiments, a bacterial strain of thespecies Megasphaera massiliensis is for use in treating or preventingprostate cancer. In certain embodiments, a bacterial strain of thespecies Megasphaera massiliensis is for use in reducing tumour size,reducing tumour growth, or reducing angiogenesis in the treatment ofprostate cancer.

In preferred embodiments, the compositions of the invention are for usein treating or preventing haematological malignancies. In certainembodiments, the compositions of the invention are for use in reducingtumour size, reducing tumour growth, or reducing angiogenesis in thetreatment of haematological malignancies. In preferred embodiments, abacterial strain of the species Megasphaera massiliensis is for use intreating or preventing haematological malignancies. In certainembodiments, a bacterial strain of the species Megasphaera massiliensisis for use in reducing tumour size, reducing tumour growth, or reducingangiogenesis in the treatment of haematological malignancies. In certainembodiments the haematological malignancy is acute leukemia. In certainembodiments the haematological malignancy is chronic leukemia. Incertain embodiments the haematological malignancy is acute myelogenousleukemia. In certain embodiments the haematological malignancy ischronic myelogenous leukemia. In certain embodiments the haematologicalmalignancy is acute lymphocytic leukemia. In certain embodiments thehaematological malignancy is chronic lymphocytic leukemia. In certainembodiments the haematological malignancy is lymphoma. In certainembodiments the haematological malignancy is multiple myeloma. Incertain embodiments the haematological malignancy is a myelodysplasticsyndrome.

In preferred embodiments, the compositions of the invention are for usein treating or preventing chronic lymphocyte leukemia. In certainembodiments, the compositions of the invention are for use in reducingtumour size, reducing tumour growth, or reducing angiogenesis in thetreatment of chronic lymphocyte leukemia. In preferred embodiments, abacterial strain of the species Megasphaera massiliensis is for use intreating or preventing chronic lymphocyte leukemia. In certainembodiments, a bacterial strain of the species Megasphaera massiliensisis for use in reducing tumour size, reducing tumour growth, or reducingangiogenesis in the treatment of chronic lymphocyte leukemia.

In preferred embodiments, the compositions of the invention are for usein treating or preventing lymphoma. In certain embodiments, thecompositions of the invention are for use in reducing tumour size,reducing tumour growth, or reducing angiogenesis in the treatment oflymphoma. In preferred embodiments, a bacterial strain of the speciesMegasphaera massiliensis is for use in treating or preventing lymphoma.In certain embodiments, a bacterial strain of the species Megasphaeramassiliensis is for use in reducing tumour size, reducing tumour growth,or reducing angiogenesis in the treatment of lymphoma. In certainembodiments the lymphoma is Hodgkin's lymphoma. In certain embodimentsthe lymphoma is Non-Hodgkin's lymphoma.

In preferred embodiments, the compositions of the invention are for usein treating or preventing gastric cancer. In certain embodiments, thecompositions of the invention are for use in reducing tumour size,reducing tumour growth, or reducing angiogenesis in the treatment ofgastric cancer. In preferred embodiments, a bacterial strain of thespecies Megasphaera massiliensis is for use in treating or preventinggastric cancer. In certain embodiments, a bacterial strain of thespecies Megasphaera massiliensis is for use in reducing tumour size,reducing tumour growth, or reducing angiogenesis in the treatment ofgastric cancer.

In certain embodiments, the compositions of the invention are for use intreating or preventing colon cancer. In certain embodiments, thecompositions of the invention are for use in treating or preventingcolorectal cancer. The compositions of the invention may have an effecton colon cancer cells and may be effective for treating colon cancer.The compositions of the invention may have an effect on colon cancercells and may be effective for treating colorectal cancer. In certainembodiments, the compositions of the invention are for use in reducingtumour size, reducing tumour growth, or reducing angiogenesis in thetreatment of colon cancer. In certain embodiments, the compositions ofthe invention are for use in reducing tumour size, reducing tumourgrowth, or reducing angiogenesis in the treatment of colorectal cancer.In certain embodiments, a bacterial strain of the species Megasphaeramassiliensis is for use in treating or preventing colon cancer. Incertain embodiments, a bacterial strain of the species Megasphaeramassiliensis is for use in treating or preventing colorectal cancer. Incertain embodiments, a bacterial strain of the species Megasphaeramassiliensis is for use in reducing tumour size, reducing tumour growth,or reducing angiogenesis in the treatment of colon cancer. In certainembodiments, a bacterial strain of the species Megasphaera massiliensisis for use in reducing tumour size, reducing tumour growth, or reducingangiogenesis in the treatment of colorectal cancer. In preferredembodiments the cancer is colorectal adenocarcinoma.

In certain embodiments, the therapeutic combinations of the inventionare for use in treating or preventing kidney cancer (also referred toherein as renal cancer). In certain embodiments, the therapeuticcombinations of the invention are for use in reducing tumour size,reducing tumour growth, or reducing angiogenesis in the treatment ofrenal cancer. In certain embodiments, a bacterial strain of the speciesMegasphaera massiliensis is for use in treating or preventing kidneycancer (also referred to herein as renal cancer). In certainembodiments, a bacterial strain of the species Megasphaera massiliensisis for use in reducing tumour size, reducing tumour growth, or reducingangiogenesis in the treatment of renal cancer. The Examples demonstratethat the therapeutic combinations of the invention have an effect onrenal cancer cells and may be effective for treating renal cancer. Inpreferred embodiments the cancer is renal cell carcinoma or transitionalcell carcinoma. In some embodiments, the cancer is of the intestine. Insome embodiments, the cancer is of a part of the body which is not theintestine. In some embodiments, the cancer is not cancer of theintestine. In some embodiments, the cancer is not colorectal cancer. Insome embodiments, the cancer is not cancer of the small intestine. Insome embodiments, the treating or preventing occurs at a site other thanat the intestine. In some embodiments, the treating or preventing occursat the intestine and also at a site other than at the intestine.

In certain embodiments, the compositions of the invention are for use intreating or preventing carcinoma. The compositions of the invention maybe effective for treating numerous types of carcinoma. In certainembodiments, the compositions of the invention are for use in treatingor preventing non-immunogenic cancer. In certain embodiments, abacterial strain of the species Megasphaera massiliensis is for use intreating or preventing carcinoma. A bacterial strain of the speciesMegasphaera massiliensis may be effective for treating numerous types ofcarcinoma. In certain embodiments, a bacterial strain of the speciesMegasphaera massiliensis is for use in treating or preventingnon-immunogenic cancer. The Examples demonstrate that the compositionsof the invention may be effective for treating non-immunogenic cancers.

The therapeutic effects of the compositions of the invention on cancermay be mediated by a pro-inflammatory mechanism. The expression of anumber of pro-inflammatory cytokines may be increased followingadministration of MRx0029. Inflammation can have a cancer-suppressiveeffect [66] and pro-inflammatory cytokines such as TNF-α are beinginvestigated as cancer therapies [67]. The up-regulation of genes suchas TNF-α shown in the Examples may indicate that the compositions of theinvention may be useful for treating cancer via a similar mechanism. Theup-regulation of a CXCR3 ligand such as CXCL9) may indicate that thecompositions of the invention elicit an IFNγ-type response. IFNγ is apotent macrophage-activating factor that can stimulate tumoricidalactivity [68], and CXCL9, for example, also has anti-cancer effects[69-71]. The Examples demonstrate that the expression of a number ofpro-inflammatory cytokines may be increased following administration ofMRx0029. Therefore, in certain embodiments, the compositions of theinvention are for use in promoting inflammation in the treatment ofcancer. In preferred embodiments, the compositions of the invention arefor use in promoting Th1 inflammation in the treatment of cancer. Th1cells produce IFNγ and have potent anti-cancer effects [66]. In certainembodiments, the compositions of the invention are for use in treatingan early-stage cancer, such as a cancer that has not metastasized, or astage 0 or stage 1 cancer. Promoting inflammation may be more effectiveagainst early-stage cancers [66]. In certain embodiments, thecompositions of the invention are for enhancing the effect of a secondanti-cancer agent. In certain embodiments, the compositions of theinvention are for use in promoting inflammation to enhance the effect ofa second anti-cancer agent. In certain embodiments, the treatment orprevention of cancer comprises increasing the level of expression of oneor more cytokines. In certain embodiments, the treatment or preventionof cancer comprises increasing the level of expression of one or morepro-inflammatory cytokines. For example, in certain embodiments, thetreatment or prevention of cancer comprises increasing the level ofexpression of one or more of IL-1β, IL-6, MIP-3α, CXCL9, IL-23, MCP-1,GMCSF and TNF-α. In certain embodiments, the treatment or prevention ofcancer comprises increasing the level of expression of one or more ofIL-1β and MIP-3α. Increases in levels of expression of any of IL-1β,IL-6 and TNF-α are known to be indicative of efficacy in treatment ofcancer.

When a bacterial strain as described herein is used in combination withlipopolysaccharide (LPS), there may be a synergistic increase in IL-1β.LPS is known to elicit a pro-inflammatory effect. Thus, in certainembodiments, the treatment or prevention comprises using a bacterialstrain as described herein in combination with an agent that upregulatesIL-1β. In certain embodiments, the treatment or prevention comprisesusing a bacterial strain as described herein in combination with LPS.Accordingly, a composition of the invention may additionally comprise anagent that upregulates IL-1β. Accordingly, a composition of theinvention may additionally comprise LPS.

In certain embodiments, the compositions of the invention are for use intreating a patient that has previously received chemotherapy. In certainembodiments, the compositions of the invention are for use in treating apatient that has not tolerated a chemotherapy treatment. Thecompositions of the invention may be particularly suitable for suchpatients.

In certain embodiments, the compositions of the invention are forpreventing relapse. The compositions of the invention may be suitablefor long-term administration. In certain embodiments, the compositionsof the invention are for use in preventing progression of cancer.

In certain embodiments, the compositions of the invention are for use intreating non-small-cell lung carcinoma. In certain embodiments, thecompositions of the invention are for use in treating small-cell lungcarcinoma. In certain embodiments, the compositions of the invention arefor use in treating squamous-cell carcinoma. In certain embodiments, thecompositions of the invention are for use in treating adenocarcinoma. Incertain embodiments, the compositions of the invention are for use intreating glandular tumors, carcinoid tumors, or undifferentiatedcarcinomas.

In certain embodiments, the compositions of the invention are for use intreating hepatoblastoma, cholangiocarcinoma, cholangiocellularcystadenocarcinoma or liver cancer resulting from a viral infection.

In certain embodiments, the compositions of the invention are for use intreating invasive ductal carcinoma, ductal carcinoma in situ or invasivelobular carcinoma.

In further embodiments, the compositions of the invention are for use intreating or preventing acute lymphoblastic leukemia (ALL), acute myeloidleukemia, adrenocortical carcinoma, basal-cell carcinoma, bile ductcancer, bladder cancer, bone tumor, osteosarcoma/malignant fibroushistiocytoma, brainstem glioma, brain tumor, cerebellar astrocytoma,cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma,supratentorial primitive neuroectodermal tumors, breast cancer,bronchial adenomas/carcinoids, Burkitt's lymphoma, carcinoid tumor,cervical cancer, chronic lymphocytic leukemia, chronic myelogenousleukemia, chronic myeloproliferative disorders, colon cancer, cutaneousT-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer,Ewing's sarcoma, intraocular melanoma, retinoblastoma, gallbladdercancer, gastric cancer, gastrointestinal carcinoid tumor,gastrointestinal stromal tumor (GIST), germ cell tumor, glioma,childhood visual pathway and hypothalamic, Hodgkin lymphoma, melanoma,islet cell carcinoma, Kaposi sarcoma, renal cell cancer, laryngealcancer, leukaemias, lymphomas, mesothelioma, neuroblastoma, non-Hodgkinlymphoma, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreaticcancer, parathyroid cancer, pharyngeal cancer, pituitary adenoma, plasmacell neoplasia, prostate cancer, renal cell carcinoma, retinoblastoma,sarcoma, testicular cancer, thyroid cancer, or uterine cancer. Infurther embodiments, the compositions of the invention are for use intreating or preventing haematologic malignancies, multiple myeloma, ormyelodysplastic syndromes.

The compositions of the invention may be particularly effective whenused in combination with further therapeutic agents. Theimmune-modulatory effects of the compositions of the invention may beeffective when combined with more direct anti-cancer agents. Therefore,in certain embodiments, the invention provides a composition comprisingthe bacterial strain of the species Megasphaera massiliensis and ananticancer agent. In certain embodiments, the composition of theinvention comprising the bacterial strain of the species Megasphaeramassiliensis is for use in stimulating a cancer to enhance itssusceptibility to treatment with a second anti-cancer agent. In certainembodiments, the composition of the invention comprising the bacterialstrain of the species Megasphaera massiliensis is for use in treating acancer, such as a brain cancer, by enhancing its susceptibility totreatment with a second anti-cancer agent. The second antic-cancer agentmay be administered concurrently, or may be administered after thecomposition comprising the bacterial strain of the species Megasphaeramassiliensis, such as at least a day, a week, or a month after.

In preferred embodiments the anticancer agent is an immune checkpointinhibitor, a targeted antibody immunotherapy, a CAR-T cell therapy, anoncolytic virus, or a cytostatic drug. In preferred embodiments, thecomposition comprises an anti-cancer agent selected from the groupconsisting of: Yervoy (ipilimumab, BMS); Keytruda (pembrolizumab,Merck); Opdivo (nivolumab, BMS); MEDI4736 (AZ/MedImmune); MPDL3280A(Roche/Genentech); Tremelimumab (AZ/MedImmune); CT-011 (pidilizumab,CureTech); BMS-986015 (lirilumab, BMS); MEDI0680 (AZ/MedImmune);MSB-0010718C (Merck); PF-05082566 (Pfizer); MEDI6469 (AZ/MedImmune);BMS-986016 (BMS); BMS-663513 (urelumab, BMS); IMP321 (Prima Biomed);LAG525 (Novartis); ARGX-110 (arGEN-X); PF-05082466 (Pfizer); CDX-1127(varlilumab; CellDex Therapeutics); TRX-518 (GITR Inc.); MK-4166(Merck); JTX-2011 (Jounce Therapeutics); ARGX-115 (arGEN-X); NLG-9189(indoximod, NewLink Genetics); INCB024360 (Incyte); IPH2201 (InnateImmotherapeutics/AZ); NLG-919 (NewLink Genetics); anti-VISTA (JnJ);Epacadostat (INCB24360, Incyte); F001287 (Flexus/BMS); CP 870893(University of Pennsylvania); MGA271 (Macrogenix); Emactuzumab(Roche/Genentech); Galunisertib (Eli Lilly); Ulocuplumab (BMS);BKT140/BL8040 (Biokine Therapeutics); Bavituximab (PeregrinePharmaceuticals); CC 90002 (Celgene); 852A (Pfizer); VTX-2337 (VentiRxPharmaceuticals); IMO-2055 (Hybridon, Idera Pharmaceuticals); LY2157299(Eli Lilly); EW-7197 (Ewha Women's University, Korea); Vemurafenib(Plexxikon); Dabrafenib (Genentech/GSK); BMS-777607 (BMS); BLZ945(Memorial Sloan-Kettering Cancer Centre); Unituxin (dinutuximab, UnitedTherapeutics Corporation); Blincyto (blinatumomab, Amgen); Cyramza(ramucirumab, Eli Lilly); Gazyva (obinutuzumab, Roche/Biogen); Kadcyla(ado-trastuzumab emtansine, Roche/Genentech); Perjeta (pertuzumab,Roche/Genentech); Adcetris (brentuximab vedotin, Takeda/Millennium);Arzerra (ofatumumab, GSK); Vectibix (panitumumab, Amgen); Avastin(bevacizumab, Roche/Genentech); Erbitux (cetuximab, BMS/Merck); Bexxar(tositumomab-I131, GSK); Zevalin (ibritumomab tiuxetan, Biogen); Campath(alemtuzumab, Bayer); Mylotarg (gemtuzumab ozogamicin, Pfizer);Herceptin (trastuzumab, Roche/Genentech); Rituxan (rituximab,Genentech/Biogen); volociximab (Abbvie); Enavatuzumab (Abbvie); ABT-414(Abbvie); Elotuzumab (Abbvie/BMS); ALX-0141 (Ablynx); Ozaralizumab(Ablynx); Actimab-C (Actinium); Actimab-P (Actinium); Milatuzumab-dox(Actinium); Emab-SN-38 (Actinium); Naptumonmab estafenatox (ActiveBiotech); AFM13 (Affimed); AFM11 (Affimed); AGS-16C3F (Agensys);AGS-16M8F (Agensys); AGS-22ME (Agensys); AGS-15ME (Agensys); GS-67E(Agensys); ALXN6000 (samalizumab, Alexion); ALT-836 (Altor Bioscience);ALT-801 (Altor Bioscience); ALT-803 (Altor Bioscience); AMG780 (Amgen);AMG 228 (Amgen); AMG820 (Amgen); AMG172 (Amgen); AMG595 (Amgen); AMG110(Amgen); AMG232 (adecatumumab, Amgen); AMG211 (Amgen/MedImmune);BAY20-10112 (Amgen/Bayer); Rilotumumab (Amgen); Denosumab (Amgen);AMP-514 (Amgen); MEDI575 (AZ/MedImmune); MEDI3617 (AZ/MedImmune);MEDI6383 (AZ/MedImmune); MEDI551 (AZ/MedImmune); Moxetumomab pasudotox(AZ/MedImmune); MEDI565 (AZ/MedImmune); MEDI0639 (AZ/MedImmune);MEDI0680 (AZ/MedImmune); MEDI562 (AZ/MedImmune); AV-380 (AVEO); AV203(AVEO); AV299 (AVEO); BAY79-4620 (Bayer); Anetumab ravtansine (Bayer);vantictumab (Bayer); BAY94-9343 (Bayer); Sibrotuzumab (BoehringerIngelheim); BI-836845 (Boehringer Ingelheim); B-701 (BioClin); BIIB015(Biogen); Obinutuzumab (Biogen/Genentech); BI-505 (Bioinvent); BI-1206(Bioinvent); TB-403 (Bioinvent); BT-062 (Biotest) BIL-010t (Biosceptre);MDX-1203 (BMS); MDX-1204 (BMS); Necitumumab (BMS); CAN-4 (Cantargia AB);CDX-011 (Celldex); CDX1401 (Celldex); CDX301 (Celldex); U3-1565 (DaiichiSankyo); patritumab (Daiichi Sankyo); tigatuzumab (Daiichi Sankyo);nimotuzumab (Daiichi Sankyo); DS-8895 (Daiichi Sankyo); DS-8873 (DaiichiSankyo); DS-5573 (Daiichi Sankyo); MORab-004 (Eisai); MORab-009 (Eisai);MORab-003 (Eisai); MORab-066 (Eisai); LY3012207 (Eli Lilly); LY2875358(Eli Lilly); LY2812176 (Eli Lilly); LY3012217 (Eli Lilly); LY2495655(Eli Lilly); LY3012212 (Eli Lilly); LY3012211 (Eli Lilly); LY3009806(Eli Lilly); cixutumumab (Eli Lilly); Flanvotumab (Eli Lilly); IMC-TR1(Eli Lilly); Ramucirumab (Eli Lilly); Tabalumab (Eli Lilly); Zanolimumab(Emergent Biosolution); FG-3019 (FibroGen); FPA008 (Five PrimeTherapeutics); FP-1039 (Five Prime Therapeutics); FPA144 (Five PrimeTherapeutics); catumaxomab (Fresenius Biotech); IMAB362 (Ganymed);IMAB027 (Ganymed); HuMax-CD74 (Genmab); HuMax-TFADC (Genmab); GS-5745(Gilead); GS-6624 (Gilead); OMP-21M18 (demcizumab, GSK); mapatumumab(GSK); IMGN289 (ImmunoGen); IMGN901 (ImmunoGen); IMGN853 (ImmunoGen);IMGN529 (ImmunoGen); IMMU-130 (Immunomedics); milatuzumab-dox(Immunomedics); IMMU-115 (Immunomedics); IMMU-132 (Immunomedics);IMMU-106 (Immunomedics); IMMU-102 (Immunomedics); Epratuzumab(Immunomedics); Clivatuzumab (Immunomedics); IPH41 (InnateImmunotherapeutics); Daratumumab (Janssen/Genmab); CNTO-95 (Intetumumab,Janssen); CNTO-328 (siltuximab, Janssen); KB004 (KaloBios);mogamulizumab (Kyowa Hakko Kirrin); KW-2871 (ecromeximab, Life Science);Sonepcizumab (Lpath); Margetuximab (Macrogenics); Enoblituzumab(Macrogenics); MGD006 (Macrogenics); MGF007 (Macrogenics); MK-0646(dalotuzumab, Merck); MK-3475 (Merck); Sym004 (Symphogen/Merck Serono);DI17E6 (Merck Serono); MOR208 (Morphosys); MOR202 (Morphosys); Xmab5574(Morphosys); BPC-1C (ensituximab, Precision Biologics); TAS266(Novartis); LFA102 (Novartis); BHQ880 (Novartis/Morphosys); QGE031(Novartis); HCD122 (lucatumumab, Novartis); LJM716 (Novartis); AT355(Novartis); OMP-21M18 (Demcizumab, OncoMed); OMP52M51 (Oncomed/GSK);OMP-59R5 (Oncomed/GSK); vantictumab (Oncomed/Bayer); CMC-544 (inotuzumabozogamicin, Pfizer); PF-03446962 (Pfizer); PF-04856884 (Pfizer);PSMA-ADC (Progenics); REGN1400 (Regeneron); REGN910 (nesvacumab,Regeneron/Sanofi); REGN421 (enoticumab, Regeneron/Sanofi); RG7221,RG7356, RG7155, RG7444, RG7116, RG7458, RG7598, RG7599, RG7600, RG7636,RG7450, RG7593, RG7596, DCDS3410A, RG7414 (parsatuzumab), RG7160(imgatuzumab), RG7159 (obintuzumab), RG7686, RG3638 (onartuzumab),RG7597 (Roche/Genentech); SAR307746 (Sanofi); SAR566658 (Sanofi);SAR650984 (Sanofi); SAR153192 (Sanofi); SAR3419 (Sanofi); SAR256212(Sanofi), SGN-LIV1A (lintuzumab, Seattle Genetics); SGN-CD33A (SeattleGenetics); SGN-75 (vorsetuzumab mafodotin, Seattle Genetics); SGN-19A(Seattle Genetics) SGN-CD70A (Seattle Genetics); SEA-CD40 (SeattleGenetics); ibritumomab tiuxetan (Spectrum); MLN0264 (Takeda); ganitumab(Takeda/Amgen); CEP-37250 (Teva); TB-403 (Thrombogenic); VB4-845(Viventia); Xmab2512 (Xencor); Xmab5574 (Xencor); nimotuzumab (YMBiosciences); Carlumab (Janssen); NY-ESO TCR (Adaptimmune); MAGE-A-10TCR (Adaptimmune); CTL019 (Novartis); JCAR015 (Juno Therapeutics);KTE-C19 CAR (Kite Pharma); UCART19 (Cellectis); BPX-401 (BellicumPharmaceuticals); BPX-601 (Bellicum Pharmaceuticals); ATTCK20 (UnumTherapeutics); CAR-NKG2D (Celyad); Onyx-015 (Onyx Pharmaceuticals); H101(Shanghai Sunwaybio); DNX-2401 (DNAtrix); VCN-01 (VCN Biosciences);Colo-Ad1 (PsiOxus Therapeutics); ProstAtak (Advantagene); Oncos-102(Oncos Therapeutics); CG0070 (Cold Genesys); Pexa-vac (JX-594, JennerexBiotherapeutics); GL-ONC1 (Genelux); T-VEC (Amgen); G207 (Medigene);HF10 (Takara Bio); SEPREHVIR (HSV1716, Virttu Biologics); OrienX010(OrienGene Biotechnology); Reolysin (Oncolytics Biotech); SVV-001(Neotropix); Cacatak (CVA21, Viralytics); Alimta (Eli Lilly), cisplatin,oxaliplatin, irinotecan, folinic acid, methotrexate, cyclophosphamide,5-fluorouracil, Zykadia (Novartis), Tafinlar (GSK), Xalkori (Pfizer),Iressa (AZ), Gilotrif (Boehringer Ingelheim), Tarceva (Astellas Pharma),Halaven (Eisai Pharma), Veliparib (Abbvie), AZD9291 (AZ), Alectinib(Chugai), LDK378 (Novartis), Genetespib (Synta Pharma),Tergenpumatucel-L (NewLink Genetics), GV1001 (Kael-GemVax), Tivantinib(ArQule); Cytoxan (BMS); Oncovin (Eli Lilly); Adriamycin (Pfizer);Gemzar (Eli Lilly); Xeloda (Roche); Ixempra (BMS); Abraxane (Celgene);Trelstar (Debiopharm); Taxotere (Sanofi); Nexavar (Bayer); IMMU-132(Immunomedics); E7449 (Eisai); Thermodox (Celsion); Cometriq (Exelixis);Lonsurf (Taiho Pharmaceuticals); Camptosar (Pfizer); UFT (TaihoPharmaceuticals); and TS-1 (Taiho Pharmaceuticals).

In some embodiments, the bacterial strain of Megasphaera massiliensisdeposited under accession number NCIMB 42761 is the only therapeuticallyactive agent in a composition of the invention.

The inventors have identified that bacterial strains from the genusMegasphaera may be particularly effective for treating or preventingcancer comprising oncogenic extracellular signal-related kinase (ERK)signalling. Extracellular signal-related kinase (ERK) is a downstreameffector in the mitogen-activated protein (MAP) kinase pathway, a highlyconserved signal transduction pathway found in all eukaryotes [72]. TheMAP-kinase pathway regulates processes such as cell proliferation,differentiation, survival and apoptosis, and aberrant activation of thepathway is closely linked to cancer pathogenesis. As described in theexamples, administration of compositions comprising Megasphaera strainscan inhibit ERK signalling in cancer cell lines; that is, reducecellular levels of phosphorylated ERK relative to total ERK protein. Theinventors have also identified that treatment with Megasphaera strainscan reduce the clonogenic survival of cancer cell lines comprisingoncogenic ERK signalling, in particular in melanoma and colorectalcancer cell lines. The inventors have also identified that treatmentwith Megasphaera strains can induce gene expression ofmicrotubule-associated protein 2 (MAP2), indicating particular utilityin treating metastatic cancers.

Therefore, in certain embodiments, the invention provides a compositioncomprising a bacterial strain of the genus Megasphaera, for use in amethod of treating or preventing cancer, wherein the cancer comprisesoncogenic ERK signalling.

As used herein “oncogenic ERK signalling” refers to the cancercomprising dysregulated cellular signalling, such asstimulus-independent signalling, via the MAP kinase pathway, the resultof which is overactive signalling by ERK (either the ERK1 or ERK2isoform, or both), which drives increased cancer cell proliferationand/or survival. ERK1 is active (i.e. signalling) when phosphorylated atpositions Thr202 and Tyr204. ERK 2 is active (i.e. signalling) whenphosphorylated at positions Thr173 and Tyr185. Accordingly, “oncogenicERK signalling” can result from the presence of oncogenic mutations in(gain of function mutations) or overexpression of positive regulators ofthe MAP kinase pathway, or oncogenic mutations in (loss of functionmutations) or downregulated expression of negative regulators of the MAPkinase pathway.

Cancer comprising oncogenic ERK signalling may alternatively be definedas cancer “exhibiting” or “characterised by” oncogenic ERK signalling.Cancer comprising oncogenic ERK signalling may alternatively be definedas cancer wherein the proliferation and/or survival of malignant cellsis “stimulated”, “induced” or “upregulated” by ERK signalling. Cancercomprising oncogenic ERK may alternatively be defined as cancercomprising, exhibiting or characterised by “stimulus-independent” ERKsignalling.

“Oncogenic mutation” encompasses any amino acid variation in a protein,relative to the wild-type protein, which promotes cancer cellproliferation and/or survival, including, but not limited to,substitutions (including single amino acid substitutions), insertionsand/or deletions. As noted above, oncogenic mutations may be loss offunction or gain of function mutations, depending on the protein and itsfunction within the MAP-kinase pathway. “Overexpression” or“downregulated expression” refer respectively to increased or decreasedexpression of a protein in a cancerous cell relative to a non-cancerouscell.

Accordingly, cancers comprising oncogenic ERK signalling include thosecomprising an oncogenic mutation in, or overexpression of, BRAF, NRAS,ARAF, CRAF, EGFR, GRB2, SOS, HRAS, KRAS4A, KRAS4B, MEK1, MEK2, ERK1 orERK2; such as BRAF, ARAF, CRAF, EGFR, GRB2, SOS, HRAS, MEK1, MEK2, ERK1or ERK2. These proteins are positive regulators of the MAP kinasepathway (i.e. oncoproteins) [72]. For example, the cancer may comprisean oncogenic mutation in BRAF, NRAS, ARAF, CRAF, EGFR, GRB2, SOS, HRAS,MEK1, MEK2, ERK1 or ERK2.

Cancers comprising oncogenic ERK signalling also include those whichcomprise (either alternatively, or in addition to, the above oncogenicmutations/overexpression) an oncogenic mutation in, or downregulatedexpression of, RSK, DUSP1, DUSP5, DUSP6 or SPRY. These proteins arenegative regulators of the MAP kinase pathway (i.e. tumour suppressorproteins) [72].

Any cancer comprising oncogenic ERK signalling can be treated orprevented using compositions of the invention, such as solid tumours orhaematological malignancies. Such cancers include, but are not limitedto, colorectal cancer, melanoma, acute lymphoblastic leukaemia (ALL),acute myeloid leukaemia, adrenocortical carcinoma, basal-cell carcinoma,bile duct cancer, bladder cancer, bone tumour, osteosarcoma/malignantfibrous histiocytoma, brainstem glioma, brain tumour, cerebellarastrocytoma, cerebral astrocytoma/malignant glioma, ependymoma,medulloblastoma, supratentorial primitive neuroectodermal tumours,breast cancer, bronchial adenomas/carcinoids, Burkitt's lymphoma,carcinoid tumour, cervical cancer, chronic lymphocytic leukaemia,chronic myelogenous leukaemia, chronic myeloproliferative disorders,cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophagealcancer, Ewing's sarcoma, intraocular melanoma, retinoblastoma,gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumour,gastrointestinal stromal tumour (GIST), germ cell tumour, glioma,childhood visual pathway and hypothalamic, Hodgkin lymphoma, islet cellcarcinoma, Kaposi sarcoma, renal cell cancer, laryngeal cancer,leukaemias, lymphomas, mesothelioma, neuroblastoma, non-Hodgkinlymphoma, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreaticcancer, parathyroid cancer, pharyngeal cancer, pituitary adenoma, plasmacell neoplasia, prostate cancer, renal cell carcinoma, retinoblastoma,sarcoma, testicular cancer, thyroid cancer, or uterine cancer.

Any cancer comprising oncogenic ERK signalling may be treated orprevented by a composition comprising a bacterial strain of the genusMegasphaera, and preferably colorectal cancer, melanoma, prostatecancer, lung adenocarcinoma such as non-small cell lung adenocarcinoma,pancreatic cancer, bladder cancer, leukaemia such as hairy cellleukaemia or acute myeloid leukaemia, glioma, pilocytic astrocytoma,ovarian cancer, papillary or follicular thyroid cancer, seminoma, livercancer, myelodysplastic syndrome, kidney cancer or Hodgkin's disease.

In preferred embodiments, the invention provides a compositioncomprising a bacterial strain of the genus Megasphaera, for use in amethod of treating or preventing cancer comprising an oncogenic mutationin BRAF, optionally wherein the cancer further comprises overexpressionof BRAF. The inventors have identified that treatment with Megasphaerastrains can inhibit the clonogenic survival, inhibit ERK signalling andupregulate MAP2 gene expression in cancer cell lines comprisingoncogenic BRAF mutations, in particular the oncogenic BRAF V600Emutation in colorectal cancer and melanoma cell lines. Therefore, inpreferred embodiments, the invention also provides a compositioncomprising a bacterial strain of the genus Megasphaera, for use in amethod of treating or preventing cancer comprising an oncogenic mutationat position 600 of BRAF, preferably BRAF V600E. In especially preferredembodiments, the cancer is colorectal cancer or melanoma.

In addition to, or instead of, an oncogenic mutation at position 600 ofBRAF (such as V600E), the cancer may comprise an oncogenic mutationselected from BRAF K601E, G469A, G469V, L597R, K601N, G464V, N581S,L597Q, A598V, G464R, G466A or G469E; optionally wherein the cancer iscolorectal cancer. In another embodiment, in addition to, or instead of,the V600E mutation, the cancer may comprise an oncogenic mutationselected from BRAF V600K, V600R or V600D; optionally wherein the canceris melanoma.

In a further aspect, the invention also provides a compositioncomprising a bacterial strain of the species Megasphaera massiliensis,for use in a method of treating colorectal cancer, such as metastaticcolorectal cancer. As shown in the examples, the inventors have foundthat Megasphaera massiliensis strains can inhibit clonogenic survivaland ERK signalling in colorectal cancer cell lines.

In a further aspect, the invention also provides a compositioncomprising a bacterial strain of the species Megasphaera massiliensis,for use in a method of treating melanoma, such as metastatic melanoma.As shown in the examples, the inventors have found that Megasphaeramassiliensis strains can inhibit clonogenic survival and ERK signallingin melanoma cell lines. Furthermore, the ability of Megasphaeramassiliensis strains to induce MAP2 gene expression in melanoma celllines indicates particular efficacy against metastatic melanoma.

In preferred embodiments, a BRAF inhibitor is administeredsimultaneously, separately or sequentially, relative to administrationof the composition comprising a bacterial strain of the genusMegasphaera. Preferably, the BRAF inhibitor is a selective inhibitor ofBRAF^(V600E), preferably selected from Vemurafenib, Dabrafenib orEncorafenib. More preferably, the BRAF inhibitor is Vemurafenib.

In a further aspect, the invention, the invention also provides acomposition comprising a bacterial strain of the genus Megasphaera and aBRAF inhibitor, preferably those defined above, for simultaneous,separate or sequential use in the treatment or prevention of cancer.

In other preferred embodiments, a cytidine analogue is administeredsimultaneously, separately or sequentially, relative to administrationof the composition comprising a bacterial strain of the genusMegasphaera. Preferably, the cytidine analogue is selected fromAzacytidine-c, Decitabine, or Zebularine. More preferably, the cytidineanalogue is Azacytidine-c.

In a further aspect, the invention also provides a compositioncomprising a bacterial strain of the genus Megasphaera and a cytidineanalogue, preferably those defined above, for simultaneous, separate orsequential use in the treatment or prevention of cancer.

In other preferred embodiments, a tubulin polymerisation inhibitor ortubulin depolymerisation inhibitor is administered simultaneously,separately or sequentially, relative to administration of thecomposition comprising a bacterial strain of the genus Megasphaera.Preferably, the tubulin polymerisation inhibitor or tubulindepolymerisation inhibitor is selected from Paclitaxel, Abraxane,Docetaxel, Epothilone, (+)-Discodermolide, Colchicine, Combretastatin,2-Methoxyestradiol, E7010, Vincristine, Vinblastine, Vinorelbine orVinflunine; more preferably Paclitaxel.

In a further aspect, the invention also provides a compositioncomprising a bacterial strain of the genus Megasphaera and a tubulinpolymerisation inhibitor or tubulin depolymerisation inhibitor,preferably those defined above, for simultaneous, separate or sequentialuse in the treatment or prevention of cancer. In a further aspect, theinvention also provides a composition comprising a bacterial strain ofthe genus Megasphaera, for use in cancer therapy by increasing thesusceptibility of the cancer to a tubulin polymerisation ordepolymerisation inhibitor, preferably those defined above.

In further such embodiments, the invention provides:

-   -   1. A composition comprising a bacterial strain of the genus        Megasphaera, for use in a method of treating or preventing        cancer, in particular, wherein the cancer comprises oncogenic        ERK signalling.    -   2. A composition for use according to embodiment 1, wherein the        cancer comprises an oncogenic mutation in, or overexpression of,        BRAF, NRAS, ARAF, CRAF, EGFR, GRB2, SOS, HRAS, KRAS4A, KRAS4B,        MEK1, MEK2, ERK1 or ERK2.    -   3. A composition for use according to any preceding embodiment,        wherein the cancer comprises an oncogenic mutation in, or        downregulated expression of, RSK, DUSP1, DUSP5, DUSP6 or SPRY.    -   4. A composition for use according to embodiment 2, wherein the        cancer comprises an oncogenic mutation in, or overexpression of,        BRAF, ARAF, CRAF, EGFR, GRB2, SOS, HRAS, MEK1, MEK2, ERK1 or        ERK2.    -   5. A composition for use according to embodiment 2, wherein the        cancer comprises an oncogenic mutation in BRAF, NRAS, ARAF,        CRAF, EGFR, GRB2, SOS, HRAS, MEK1, MEK2, ERK1 or ERK2.    -   6. The composition for use according to any preceding        embodiment, wherein the cancer comprises an oncogenic mutation        in BRAF or NRAS, optionally wherein the cancer further comprises        overexpression of BRAF or NRAS.    -   7. The composition for use according to embodiment 6, wherein        the cancer comprises an oncogenic mutation in BRAF, optionally        wherein the cancer further comprises overexpression of BRAF.    -   8. The composition for use according to embodiment 7, wherein        the cancer comprises an oncogenic mutation at position 600 of        BRAF.    -   9. The composition for use according to any of embodiments 6-8,        wherein the cancer comprises an oncogenic mutation selected from        BRAF V600E, K601E, G469A, G469V, L597R, K601N, G464V, N581S,        L597Q, A598V, G464R, G466A or G469E; optionally wherein the        cancer is colorectal cancer.    -   10. The composition for use according to any of embodiments 6-8,        wherein the cancer comprises an oncogenic mutation selected from        BRAF V600E, V600K, V600R or V600D; optionally wherein the cancer        is melanoma.    -   11. The composition for use according to any of embodiments        6-10, wherein the cancer comprises the oncogenic mutation BRAF        V600E.    -   12. The composition for use according to any of embodiments        6-11, wherein the cancer comprises the oncogenic mutation NRAS        Q61R, optionally wherein the cancer is melanoma.    -   13. The composition for use according to any preceding        embodiment, wherein the cancer is selected from colorectal        cancer, melanoma, prostate cancer, lung adenocarcinoma such as        non-small cell lung adenocarcinoma, pancreatic cancer, bladder        cancer, leukaemia such as hairy cell leukaemia or acute myeloid        leukaemia, glioma, pilocytic astrocytoma, ovarian cancer,        papillary or follicular thyroid cancer, seminoma, liver cancer,        myelodysplastic syndrome, kidney cancer or Hodgkin's disease.    -   14. The composition for use according to any preceding        embodiment, wherein the cancer is colorectal cancer.    -   15. The composition for use according to any of embodiments        1-13, wherein the cancer is melanoma.    -   16. The composition for use according to any preceding        embodiment, wherein the bacterial strain is of the species        Megasphaera massiliensis.    -   17. The composition for use according to any preceding        embodiment, in a method of inhibiting ERK1 and/or ERK2        signalling in the treatment or prevention of the cancer.    -   18. The composition for use according to any preceding        embodiment, in a method of inhibiting ERK1 and/or ERK2        phosphorylation in the treatment or prevention of the cancer.    -   19. The composition for use according to any preceding        embodiment, in a method of inducing MAP2 gene expression in the        treatment or prevention of the cancer.    -   20. The composition for use according to any preceding        embodiment, in a method of reducing tumour size, tumour growth,        preventing or inhibiting metastasis, or preventing angiogenesis        in the treatment or prevention of the cancer.    -   21. The composition for use according to any preceding        embodiment, in a method of inhibiting metastasis in the        treatment of the cancer.    -   22. The composition for use according to any preceding        embodiment, wherein the method comprises simultaneous, separate        or sequential administration of a BRAF inhibitor, relative to        administration of the composition.    -   23. The composition for use according to embodiment 22, wherein        the BRAF selectively inhibits BRAF^(V600E), preferably wherein        the BRAF inhibitor is selected from Vemurafenib, Dabrafenib or        Encorafenib.    -   24. The composition for use according to embodiment 23, wherein        the BRAF inhibitor is Vemurafenib.    -   25. The composition for use according to any preceding        embodiment, wherein the method comprises simultaneous, separate        or sequential administration of a cytidine analogue, relative to        administration of the composition.    -   26. The composition for use according to embodiment 25, wherein        the cytidine analogue is selected from Azacytidine-c,        Decitabine, or Zebularine.    -   27. The composition for use according to embodiment 26, wherein        the cytidine analogue is Azacytidine-c.    -   28. The composition for use according to any preceding        embodiment, wherein the method comprises simultaneous, separate        or sequential administration of a tubulin polymerisation        inhibitor or tubulin depolymerisation inhibitor, relative to        administration of the composition.    -   29. The composition for use according to embodiment 28, wherein        the tubulin polymerisation inhibitor or tubulin depolymerisation        inhibitor is selected from Paclitaxel, Abraxane, Docetaxel,        Epothilone, (+)-Discodermolide, Colchicine, Combretastatin,        2-Methoxyestradiol, E7010, Vincristine, Vinblastine, Vinorelbine        or Vinflunine.

Preferably, cancers comprising oncogenic ERK signalling which can betreated or prevented using compositions of the invention (in particular,those comprising a bacterial strain of the species Megasphaeramassiliensis) include, but are not limited to, colorectal cancer,melanoma, prostate cancer, lung adenocarcinoma such as non-small celllung adenocarcinoma, pancreatic cancer, bladder cancer, leukaemia suchas hairy cell leukaemia or acute myeloid leukaemia, glioma, pilocyticastrocytoma, ovarian cancer, papillary or follicular thyroid cancer,seminoma, liver cancer, myelodysplastic syndrome, kidney cancer andHodgkin's disease. Such cancers have been reported as comprising anoveractive MAP-kinase pathway (i.e. oncogenic ERK signalling) [72].

In a particular embodiment, compositions of the invention are for use intreating or preventing cancer comprising an oncogenic mutation in BRAFor NRAS, optionally wherein the cancer further comprises overexpressionof BRAF or NRAS. Preferably, compositions of the invention (inparticular, those comprising a bacterial strain of the speciesMegasphaera massiliensis) are for use in treating or preventing cancerwhich comprises an oncogenic mutation in BRAF, and optionaloverexpression of BRAF.

Oncogenic mutations in BRAF include V600E, K601E, G469A, G469V, L597R,K601N, G464V, N581S, L597Q, A598V, G464R, G466A or G469E, which havebeen identified in colorectal cancers [73], and compositions of theinvention compositions (in particular, those comprising a bacterialstrain of the species Megasphaera massiliensis) may be used to treat orprevent such cancers. Further oncogenic mutations in BRAF include V600E,V600K, V600R or V600D, which have been identified in melanomas [74], andcompositions of the invention compositions (in particular, thosecomprising a bacterial strain of the species Megasphaera massiliensis)may be used to treat or prevent such cancers. Amino acids in BRAF arenumbered according to UniProt entry P15056 [75] (wild-type BRAF).

In an especially preferred embodiment, compositions of the invention (inparticular, those comprising a bacterial strain of the speciesMegasphaera massiliensis) are for use in treating or preventing cancerwhich comprises the mutation BRAF V600E. The cancer cell lines SKMEL28,451Lu and HT29 comprise this mutation in BRAF, and a strain ofMegasphaera was found in the Examples to inhibit clonogenic survival,inhibit ERK signalling and induce MAP2 gene expression in such celllines. The cancer may further comprise the oncogenic mutation NRAS Q61R.The cancer cell line SKMEL2 comprises this mutation in NRAS, and astrain of Megasphaera was found in the Examples to induce MAP2 geneexpression in this cell line.

The HT29 cell line used in the Examples is a colorectal cancer cellline, and a strain of Megasphaera was found to inhibit clonogenicsurvival and inhibit ERK signalling in this cell line. Therefore, inespecially preferred embodiments, compositions of the invention (inparticular, those comprising a bacterial strain of the speciesMegasphaera massiliensis) are used to treat or prevent colorectalcancer, such as colorectal cancer which comprises the mutation BRAFV600E.

The SKMEL2 and SKMEL28 and 451Lu cell lines used in the Examples aremelanoma cell lines, and a strain of Megasphaera was found to inhibitclonogenic survival, inhibit ERK signalling and induce MAP2 geneexpression in such cell lines. Therefore, in especially preferredembodiments, compositions of the invention (in particular, thosecomprising a bacterial strain of the species Megasphaera massiliensis)are used to treat or prevent melanoma, such as melanoma which comprisesthe mutation BRAF V600E.

In another aspect, the composition of the invention comprises abacterial strain of the species Megasphaera massiliensis, for use in amethod of treating colorectal cancer. In another aspect, the compositionof the invention comprises a bacterial strain of the species Megasphaeramassiliensis, for use in a method of treating melanoma.

In any of the aspects and embodiments detailed above, the composition ofthe invention (in particular, a composition comprising a bacterialstrain of the species Megasphaera massiliensis) is preferably for use intreating a metastatic cancer. As reported in the Examples, a strain ofMegasphaera was found to upregulated MAP2 gene expression. MAP2 has beenfound to be highly expressed in primary cutaneous melanomas, but hasreduced expression in metastatic melanomas [76]. It has been proposedthat increased expression of microtubule-stabilizing proteins ortreatment with microtubule stabilizing proteins such as MAP2 mayinterfere with the dynamic instability of microtubules which is requiredduring cell division. Therefore, upregulation of MAP2 is thought tohamper cell division and delay tumour growth in cancer [76], indicatingthat compositions of the invention may have particular use in treatingmetastatic cancers.

As demonstrated in the Examples, compositions of the inventioncomprising a Megasphaera strain have the effects of inducing MAP2 geneexpression and inhibiting ERK signalling in melanoma and colorectalcancer cell lines. Therefore, compositions of the invention are usefulin methods of inhibiting ERK signalling, such as ERK1 and/or ERK2signalling, in the treatment or prevention of cancers comprisingoncogenic ERK signalling, as defined above. Compositions of theinvention are also useful in methods of inhibiting ERK phosphorylation,such as ERK1 and/or ERK2 phosphorylation, in the treatment or preventionof such cancers. Compositions of the invention are also useful inmethods of inducing MAP2 gene expression in the treatment or preventionof such cancers. MAP2 gene expression has been associated with increasedcancer sensitivity to microtubule-targeting compounds such as Paclitaxel[77]. Therefore, compositions of the invention may be used to increasethe susceptibility of such cancers to a tubulin polymerisation ordepolymerisation inhibitor, in particular Paclitaxel. Compositions ofthe invention are also useful in methods of reducing tumour size,reducing tumour growth, preventing or inhibiting metastasis, orpreventing angiogenesis in the treatment or prevention of cancerscomprising oncogenic ERK signalling. Due to the effects on MAP2 geneexpression demonstrated in the Examples, compositions of the inventionare preferably for use in methods of inhibiting metastasis in thetreatment of such cancers.

In a further aspect, a composition comprising a bacterial strain of thegenus Megasphaera is for use in a method of inhibiting ERK1 and/or ERK2signalling in the treatment or prevention of cancer. In a furtheraspect, a composition comprising a bacterial strain of the genusMegasphaera is for use in a method of inhibiting ERK1 and/or ERK2phosphorylation in the treatment or prevention of cancer. In a furtheraspect, a composition comprising a bacterial strain of the genusMegasphaera is for use in a method of inducing MAP2 gene expression inthe treatment or prevention of cancer. In said further aspects,preferably the cancers are characterised as detailed above (“Cancers andcharacteristics thereof”).

In certain embodiments, the composition of the invention is for use intreating small bowel cancer, such as small bowel adenocarcinoma. Themethotrexate-treated HT29 cell line used in the examples has a phenotyperesembling epithelial cells of the small intestine and the compositionsof the invention were shown to have a useful effect on such cells. Incertain embodiments, the compositions of the invention are used topromote apoptosis in the treatment or prevention of cancer, inparticular of small bowel cancer.

In certain embodiments, the composition of the invention is for use in amethod of inducing GPR109a gene expression in the treatment orprevention of cancer.

In certain embodiments, the composition of the invention is for use in amethod of increasing IL-8 levels in the treatment or prevention ofcancer.

In certain embodiments, the composition of the invention is for use intreating colorectal cancer, such as colorectal adenocarcinoma. TheCaco-2 cell line used in the examples is a colorectal adenocarcinomacell line and the compositions of the invention were shown to have auseful effect on such cells.

In certain embodiments, the compositions are for use in treating orpreventing metastatic melanoma, small cell lung cancer or adenosquamouslung carcinoma. The effect on NSE shown in the examples suggests thatthe compositions of the invention may be particular effective againstthese cancers.

In certain embodiments, the composition of the invention is not for usein treating cancer. In certain embodiments, the composition of theinvention is for use in treating a disease or disorder that is notcancer.

Use as a Vaccine Adjuvant

The Examples show that administration of the compositions of theinvention can lead to an increase in expression of Tumour NecrosisFactor alpha (TNF-α). TNF-α is known to be important for vaccineresponses. For example, TNF-α has been shown to be required for anefficient vaccine response in a flu vaccination of the elderlypopulation [78]. Since administration of the compositions of theinvention were shown to increase TNF-α expression, compositions of theinvention may be useful as a vaccine adjuvant. In one embodiment, thecompositions of the invention are for use as a vaccine adjuvant byincreasing the level and/or activity of TNF-α. In one embodiment, thecompositions of the invention are for use as a vaccine adjuvant. In oneembodiment, the compositions of the invention are for use as a vaccineadjuvant in influenza therapy. In certain embodiments, the compositionsof the invention are for use in enhancing an immune response against anantigen. In certain embodiments, the invention provides a composition tobe administered in combination with an antigen. In certain embodiments,the compositions of the invention are for administration to a patientshortly prior to or after vaccination.

The Examples also show that administration of the compositions of theinvention can lead to an increase in expression of IL-6. Increased IL-6expression has been associated with vaccine responses for many diseases.For example, IL-6 was produced by CD14+CD16− inflammatory monocytesafter adults were administered an influenza vaccine [79], and higherlevels of IL-6 were associated with achieving a vaccine response to aninfluenza vaccine [80]. Furthermore, IL-6 was produced after injectionof the AS03 adjuvant system [81] and downregulation of IL-6 in mice wasshown to reduce the helper T cell response after administration of atuberculosis vaccine [82]. Since administration of the compositions ofthe invention were shown to increase IL-6 expression, compositions ofthe invention may be useful as a vaccine adjuvant. In one embodiment,the compositions of the invention are for use as a vaccine adjuvant byincreasing the level and/or activity of IL-6. In one embodiment, thecompositions of the invention are for use as a vaccine adjuvant. In oneembodiment, the compositions of the invention are for use as a vaccineadjuvant in tuberculosis therapy.

Furthermore, IL-6 and TNF-α expression have been shown to be correlatedwith the efficacy of a therapeutic HIV vaccine [Huang et al] atuberculosis vaccine and a chlamydia vaccine [83]. Su et al. [84] showedthat co-inoculation of IL-6 or TNF-α with the FMDV DNA vaccine resultedin increased IFN-γ expression by CD4+ and CD8+ T cells, higherexpression of IL-4 in CD4+ T cells and a higher antigen-specificcytotoxic response. Since administration of the compositions of theinvention were shown to increase IL-6 and TNF-α expression, compositionsof the invention may be useful as a vaccine adjuvant. In one embodiment,the compositions of the invention may be useful as a vaccine adjuvant byincreasing the level and/or activity of TNF-α. In one embodiment, thecompositions of the invention may be useful as a vaccine adjuvant byincreasing the level and/or activity of IL-6. In a particularembodiment, the compositions of the invention may be useful as a vaccineadjuvant by increasing the level and/or activity of TNF-α and IL-6. Inone embodiment, the compositions of the invention are for use as avaccine adjuvant in HIV therapy. In one embodiment, the compositions ofthe invention are for use as a vaccine adjuvant in chlamydia therapy.

The Examples show that administration of the compositions of theinvention can lead to an increase in expression of MIP-3α. MIP-3α hasbeen shown to increase the response to a HIV vaccine [85]. Sinceadministration of the compositions of the invention were shown toincrease MIP-3α expression, compositions of the invention may be usefulas a vaccine adjuvant.

The Examples also show that administration of the compositions of theinvention can lead to an increase in expression of IL-1β. Li et al. [86]showed that the adjuvant aluminium hydroxide activated the secretion ofIL-1β, and suggested that IL-β itself can act as an adjuvant. Sinceadministration of the compositions of the invention were shown toincrease IL-1β expression, compositions of the invention may be usefulas a vaccine adjuvant. The Examples show that administration of thecompositions of the invention can increase the ratio of CD8+ T cells toTregs. Adjuvants have been shown to stimulate CD8+ T cells [87] andsince administration of the compositions of the invention were shown toincrease the ratio of CD8+ T cells to Tregs, compositions of theinvention may be useful as a vaccine adjuvant. In one embodiment,compositions of the invention are for use as a vaccine adjuvant. In oneembodiment, the compositions of the invention are for use as a vaccineadjuvant by increasing the ratio of CD8+ T cells to Tregs.

The Examples also show that administration of the compositions of theinvention can lead to an increase in expression of IL-8. Increased I1-8expression has been associated with vaccine responses for many diseases.For example, higher levels of IL-8 were associated with achieving avaccine response to an avian influenza vaccine [88]. Furthermore, IL-8serves as a molecular adjuvant in a DNA vaccination model [89].Therefore, IL-8 may be used as an immunostimulant to enhance the immuneefficiency of, for example, an avian influenza vaccine. Sinceadministration of the compositions of the invention were shown toincrease IL-8 expression, compositions of the invention may be useful asa vaccine adjuvant. In one embodiment, the compositions of the inventionare for use as a vaccine adjuvant by increasing the level and/oractivity of IL-8. In one embodiment, the compositions of the inventionare for use as a vaccine adjuvant. In one embodiment, the compositionsof the invention are for use as a vaccine adjuvant in influenza therapy.In some embodiments, when used as a vaccine adjuvant, the compositionsof the invention will be administered on their own to provide anadjuvant effect for an antigen that has been separately administered tothe patient. In certain embodiments, the composition of the invention isadministered orally, whilst the antigen is injected parenterally.

The compositions of the invention may be used for enhancing an immuneresponse to any useful antigen. Exemplary antigens for use with theinvention include: viral antigens, such as viral surface proteins;bacterial antigens, such as protein and/or saccharide antigens; fungalantigens; parasite antigens; and tumor antigens. The invention isparticularly useful for vaccines against influenza virus, HIV, hookworm,hepatitis B virus, herpes simplex virus, rabies, respiratory syncytialvirus, cytomegalovirus, Staphylococcus aureus, chlamydia, SARScoronavirus, varicella zoster virus, Streptococcus pneumoniae, Neisseriameningitidis, Mycobacterium tuberculosis, Bacillus anthracis, EpsteinBarr virus, human papillomavirus, etc. Further antigens for use with theinvention include glycoprotein and lipoglycan antigens, archaeaantigens, melanoma antigen E (MAGE), Carcinoembryonic antigen (CEA),MUC-1, HER2, sialyl-Tn (STn), human telomerase reverse transcriptase(hTERT), Wilms tumour gene (WT1), CA-125, prostate-specific antigen(PSA), Epstein-Barr virus antigens, neoantigens, oncoproteins,amyloid-beta, Tau, PCSK9 and habit forming substances, for examplenicotine, alcohol or opiates.

Preferred antigens for use with the invention include pathogen antigensand tumour antigens. An antigen will elicit an immune response specificfor the antigen that will be effective for protecting against infectionwith the pathogen or attacking the tumour. Antigens may be, for example,peptides or polysaccharides.

The invention also provides the use of: (i) an aqueous preparation of anantigen; and (ii) a composition comprising a bacterial strain of thegenus Megasphaera, preferably the species Megasphaera massiliensis, inthe manufacture of a medicament for raising an immune response in apatient.

The immune response raised by these methods and uses will generallyinclude an antibody response, preferably a protective antibody response.

In some embodiments, a bacterial strain of the genus Megasphaera isengineered to present an antigen. Presenting an antigen on the bacterialstrain of the invention may maximise the immunostimulatory activitiesand further enhance the protective immune response generated against theantigen. In addition, manufacturing and delivering therapeuticscomprising an antigen and a bacteria of the invention may be moreefficient and effective this way than when each of the antigen and thecomposition comprising the bacterial strain are manufactured andadministered separately. Therefore, in some embodiments, the inventionprovides a composition comprising a bacterial strain of the genusMegasphaera that presents an antigen, for example on its cell surface.In some embodiments, the composition comprising the bacterial strainthat presents an antigen is for use as a vaccine antigen. In someembodiments, the antigen is derived from HIV, hookworm, hepatitis Bvirus, herpes simplex virus, rabies, respiratory syncytial virus,cytomegalovirus, Staphylococcus aureus, chlamydia, SARS coronavirus,varicella zoster virus, Streptococcus pneumoniae, Neisseriameningitidis, Mycobacterium tuberculosis, Bacillus anthracis, EpsteinBarr virus or human papillomavirus. In some embodiments, the antigen isa glycoprotein antigen, lipoglycan antigen, archaea antigen, melanomaantigen E (MAGE), Carcinoembryonic antigen (CEA), MUC-1, HER2, sialyl-Tn(STn), human telomerase reverse transcriptase (hTERT), Wilms tumour gene(WT1), CA-125, prostate-specific antigen (PSA), Epstein-Barr virusantigens, neoantigens, oncoproteins, amyloid-beta, Tau, PCSK9 or a habitforming substance, such as, alcohol, opiates and the like.

In some embodiments, the bacteria of the invention expresses one or moreantigens. Generally the antigen will be expressed recombinantly and willbe heterologous to the bacteria of the invention. Therefore, theinvention provides a bacterial strain of the genus Megasphaera thatexpresses a heterologous antigen. The antigen may be part of a fusionpolypeptide expressed with one or more polypeptides homologous to thebacteria. In some embodiments, the bacteria expresses the antigen as anon-fusion polypeptide. In some embodiments, the invention provides acomposition comprising a cell of a bacterial strain of the genusMegasphaera, wherein the cell expresses a heterologous antigen. In someembodiments, the composition is for use as a vaccine. In someembodiments, the invention provides a cell of a bacterial strain of thegenus Megasphaera, wherein the cell expresses a heterologous antigen. Insome embodiments, the cell is for use as a vaccine.

Exemplary antigens for use with the invention include: viral antigens,such as viral surface proteins; bacterial antigens, such as proteinand/or saccharide antigens; fungal antigens; parasite antigens; andtumor antigens. Further antigens for expressing in a bacterial strain ofthe genus Megasphaera include glycoprotein and lipoglycan antigens,archaea antigens, melanoma antigen E (MAGE), Carcinoembryonic antigen(CEA), MUC-1, HER2, sialyl-Tn (STn), human telomerase reversetranscriptase (hTERT), Wilms tumour gene (WT1), CA-125,prostate-specific antigen (PSA), Epstein-Barr virus antigens,neoantigens, oncoproteins, amyloid-beta, Tau, PCSK9 and habit formingsubstances, for example nicotine, alcohol, opiates, or the like.

The invention may also be useful for enhancing the response to vaccinesagainst non-communicable diseases such as Alzheimer's Disease and otherneurodegenerative disorders, in which case the antigen for use with theinvention may be amyloid-beta or Tau. Other such antigens fornon-communicable diseases include PCSK9 (for the treatment of elevatedcholesterol).

The invention may also be useful for enhancing the response to vaccinesagainst habit forming substances, for example nicotine, alcohol oropiates.

Cell Therapies

Chimeric Antigen Receptor T Cell (CAR-T) Therapy

The Examples also show that administration of the compositions of theinvention can lead to an increase in expression of IL-6. Increased 11-6expression has been correlated with response to CD19 CAR-T therapy ofchronic lymphocyte leukaemia. An increase in serum IL-6 was associatedwith CAR-T cell expansion, whereas inhibition of IL-6 was associatedwith inhibition of CAR-T cell proliferation [90]. Since administrationof the compositions of the invention were shown to increase IL-6expression, compositions of the invention may be useful in cell therapy,in particular CAR-T cell therapy. In one embodiment, the compositions ofthe invention are for use in cell therapy. In one embodiment, thecompositions of the invention are for use in CAR-T cell therapy. In oneembodiment, compositions of the invention are for use in the treatmentof chronic lymphocyte leukaemia.

Surprisingly, the Examples also show that administration of thecompositions of the invention selectively reduced the percentage ofTregs in a population of PBMCs (FIG. 6C). Selective depletion of Tregshas been shown to enhance the efficacy of cytotoxic lymphocytes [91].CAR-T cells are a subset of cytotoxic lymphocytes, and therefore it isthought that selective depletion of Tregs is effective in CAR-T celltherapy. Since administration of the compositions of the invention wereshown to deplete Tregs, compositions of the invention may be useful incell therapy, in particular CAR-T cell therapy.

Therefore, the compositions of the invention may be useful in celltherapy, in particular in enhancing the response to a cell therapy.

Mesenchymal Stem Cell (MSC) Therapy

Mesenchymal stem cell (MSC) therapy has been reported to haveimmunostimulatory properties. When MSCs are treated with LPS, theyupregulate pro-inflammatory cytokines IL-6 and IL-8 which causesincreased B cell proliferation [92]. Therefore, since compositions ofthe invention were shown to increase the expression of IL-6, they may beuseful in combination with MSC cell therapy.

Stem Cell Transplantation Therapy

It has been reported that, instead of using undifferentiated stem cellsin stem cell transplantation therapy, it may be beneficial todifferentiate stem cells to some extent prior to transplantation. Forexample, Heng et al. [93] reported that cardiomyogenic differentiationof stem cells may be beneficial by having a higher engraftmentefficiency, enhanced regeneration of myocytes and increased restorationof heart function. Since administration of the compositions of theinvention initiated neuronal differentiation in undifferentiatedneuroblastoma cells, compositions of the invention may be useful forstem cell differentiation in stem cell transplantation therapy.

Immunosenescence

The Examples also show that administration of the compositions of theinvention can selectively deplete Tregs and increase B cell numbers(FIG. 6C and FIG. 6F). Fulop et al. [94] identified that an increase inTreg cell number and a decrease in B cell number are associated withaging in the adaptive immune system. Therefore, compositions of theinvention may be used to prevent or delay immunosenescence. In oneembodiment, compositions of the invention are for use in preventingimmunosenescence. In another embodiment, compositions of the inventionare for use in delaying immunosenescence characterised by an increase inTreg cell number. In another embodiment, compositions of the inventionare for use in delaying immunosenescence characterised by a decrease inB cell number. In another embodiment, compositions of the invention arefor use in delaying immunosenescence characterised by an increase inTreg cell number and a decrease in B cell number. In one embodiment,compositions of the invention are for use in delaying immunosenescenceby decreasing Treg cell number. In one embodiment, compositions of theinvention are for use in delaying immunosenescence by increasing B cellnumber. In another embodiment, compositions of the invention are for usein delaying immunosenescence by decreasing Treg cell number andincreasing B cell number. In one embodiment, compositions of theinvention are for use in treating diseases caused by immunosenescence.In one embodiment, compositions of the invention are for use in treatingaging-related diseases by delaying and/or preventing immunosenescence.

Furthermore, it has been proposed that vaccine adjuvants may overcomeimmunosenescence [95]. Since the compositions of the invention aresuitable for use as a vaccine adjuvant, compositions of the inventionmay be useful for preventing or delaying immunosenescence. In anotherembodiment, compositions of the invention are for use in delaying and/orpreventing immunosenescence as a vaccine adjuvant. In anotherembodiment, compositions of the invention are for use as a vaccineadjuvant, wherein the compositions delay and/or preventimmunosenescence.

Diseases that are associated with immunosenescence includecardiovascular disease, neurodegenerative diseases, such as Alzheimer'sdisease and Parkinson's disease, cancer, diabetes mellitus type 2 [96]and autoimmune disorders [97]. In one embodiment, compositions of theinvention are for use in treating cardiovascular disease. In oneembodiment, compositions of the invention are for use in treatingcardiovascular disease by delaying and/or preventing immunosenescence.In one embodiment, compositions of the invention are for use in treatingneurodegenerative diseases. In one embodiment, compositions of theinvention are for use in treating neurodegenerative diseases inparticular Alzheimer's disease and Parkinson's disease by delayingand/or preventing immunosenescence. In one embodiment, compositions ofthe invention are for use in treating cancer by delaying and/orpreventing immunosenescence. In one embodiment, compositions of theinvention are for use in treating diabetes mellitus type 2. In oneembodiment, compositions of the invention are for use in treatingdiabetes mellitus type 2 by delaying and/or preventing immunosenescence.In one embodiment, compositions of the invention are for use in treatingautoimmune disorders. In one embodiment, compositions of the inventionare for use in treating autoimmune disorders by delaying and/orpreventing immunosenescence.

Modes of Administration

Preferably, the compositions of the invention are to be administered tothe gastrointestinal tract in order to enable delivery to and/or partialor total colonisation of the intestine with the bacterial strain of theinvention. Generally, the compositions of the invention are administeredorally, but they may be administered rectally, intranasally, or viabuccal or sublingual routes.

In certain embodiments, the compositions of the invention may beadministered as a foam, as a spray or a gel.

In certain embodiments, the compositions of the invention may beadministered as a suppository, such as a rectal suppository, for examplein the form of a Theobroma oil (cocoa butter), synthetic hard fat (e.g.suppocire, witepsol), glycero-gelatin, polyethylene glycol, or soapglycerin composition.

In certain embodiments, the composition of the invention is administeredto the gastrointestinal tract via a tube, such as a nasogastric tube,orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneousendoscopic gastrostomy (PEG), or a port, such as a chest wall port thatprovides access to the stomach, jejunum and other suitable access ports.

The compositions of the invention may be administered once, or they maybe administered sequentially as part of a treatment regimen. In certainembodiments, the compositions of the invention are to be administereddaily.

In certain embodiments of the invention, treatment according to theinvention is accompanied by assessment of the patient's gut microbiota.Treatment may be repeated if delivery of and/or partial or totalcolonisation with the strain of the invention is not achieved such thatefficacy is not observed, or treatment may be ceased if delivery and/orpartial or total colonisation is successful and efficacy is observed.

In certain embodiments, the composition of the invention may beadministered to a pregnant animal, for example a mammal such as a humanin order to reduce the likelihood of cancer developing in her child inutero and/or after it is born.

The compositions of the invention may be administered to a patient thathas been diagnosed with a disease or condition mediated reduced immuneactivity, or that has been identified as being at risk of a disease orcondition mediated by reduced immune activity. The compositions may alsobe administered as a prophylactic measure to prevent the development ofdiseases or conditions mediated by reduced immune activity in a healthypatient.

The compositions of the invention may be administered to a patient thathas been diagnosed with cancer, or that has been identified as being atrisk of a cancer. For example, the patient may have reduced or absentcolonisation by Megasphaera, and in particular Megasphaera massiliensis.

The compositions of the invention may be administered as a food product,such as a nutritional supplement.

Generally, the compositions of the invention are for the treatment ofhumans, although they may be used to treat animals including monogastricmammals such as poultry, pigs, cats, dogs, horses or rabbits. Thecompositions of the invention may be useful for enhancing the growth andperformance of animals. If administered to animals, oral gavage may beused.

Compositions

Generally, the composition of the invention comprises bacteria. Inpreferred embodiments of the invention, the composition is formulated infreeze-dried form. For example, the composition of the invention maycomprise granules or gelatin capsules, for example hard gelatincapsules, comprising a bacterial strain of the invention.

Preferably, the composition of the invention comprises lyophilisedbacteria. Lyophilisation of bacteria is a well-established procedure andrelevant guidance is available in, for example, references [98, 100].

Alternatively, the composition of the invention may comprise a live,active bacterial culture.

In preferred embodiments, the composition of the invention isencapsulated to enable delivery of the bacterial strain to theintestine. Encapsulation protects the composition from degradation untildelivery at the target location through, for example, rupturing withchemical or physical stimuli such as pressure, enzymatic activity, orphysical disintegration, which may be triggered by changes in pH. Anyappropriate encapsulation method may be used. Exemplary encapsulationtechniques include entrapment within a porous matrix, attachment oradsorption on solid carrier surfaces, self-aggregation by flocculationor with cross-linking agents, and mechanical containment behind amicroporous membrane or a microcapsule. Guidance on encapsulation thatmay be useful for preparing compositions of the invention is availablein, for example, references [101] and [102].

The composition may be administered orally and may be in the form of atablet, capsule or powder. Encapsulated products are preferred becauseMegasphaera are anaerobes. Other ingredients (such as vitamin C, forexample), may be included as oxygen scavengers and prebiotic substratesto improve the delivery and/or partial or total colonisation andsurvival in vivo. Alternatively, the probiotic composition of theinvention may be administered orally as a food or nutritional product,such as milk or whey based fermented dairy product, or as apharmaceutical product.

The composition may be formulated as a probiotic.

A composition of the invention includes a therapeutically effectiveamount of a bacterial strain of the invention. A therapeuticallyeffective amount of a bacterial strain is sufficient to exert abeneficial effect upon a patient. A therapeutically effective amount ofa bacterial strain may be sufficient to result in delivery to and/orpartial or total colonisation of the patient's intestine.

A suitable daily dose of the bacteria, for example for an adult human,may be from about 1×10³ to about 1×10¹¹ colony forming units (CFU); forexample, from about 1×10⁷ to about 1×10¹⁰ CFU; in another example fromabout 1×10⁶ to about 1×10¹⁰ CFU; in another example from about 1×10⁷ toabout 1×10¹¹ CFU; in another example from about 1×10⁸ to about 1×10¹⁰CFU; in another example from about 1×10⁸ to about 1×10¹¹ CFU.

In certain embodiments, the dose of the bacteria is at least 10⁹ cellsper day, such as at least 10¹⁰, at least 10¹¹, or at least 10¹² cellsper day.

In certain embodiments, the composition contains the bacterial strain inan amount of from about 1×10⁶ to about 1×10¹¹ CFU/g, respect to theweight of the composition; for example, from about 1×10⁸ to about 1×10¹⁰CFU/g. The dose may be, for example, 1 g, 3 g, 5 g, and 10 g.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the amount of the bacterial strain is from about1×10³ to about 1×10¹¹ colony forming units per gram with respect to aweight of the composition.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the composition is administered at a dose ofbetween 500 mg and 1000 mg, between 600 mg and 900 mg, between 700 mgand 800 mg, between 500 mg and 750 mg or between 750 mg and 1000 mg. Incertain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the lyophilised bacteria in the pharmaceuticalcomposition is administered at a dose of between 500 mg and 1000 mg,between 600 mg and 900 mg, between 700 mg and 800 mg, between 500 mg and750 mg or between 750 mg and 1000 mg.

Typically, a probiotic, such as the composition of the invention, isoptionally combined with at least one suitable prebiotic compound. Aprebiotic compound is usually a non-digestible carbohydrate such as anoligo- or polysaccharide, or a sugar alcohol, which is not degraded orabsorbed in the upper digestive tract. Known prebiotics includecommercial products such as inulin and transgalacto-oligosaccharides.

In certain embodiments, the probiotic composition of the presentinvention includes a prebiotic compound in an amount of from about 1 toabout 30% by weight, respect to the total weight composition, (e.g. from5 to 20% by weight). Carbohydrates may be selected from the groupconsisting of: fructo-oligosaccharides (or FOS), short-chainfructo-oligosaccharides, inulin, isomalt-oligosaccharides, pectins,xylo-oligosaccharides (or XOS), chitosan-oligosaccharides (or COS),beta-glucans, arable gum modified and resistant starches, polydextrose,D-tagatose, acacia fibers, carob, oats, and citrus fibers. In oneaspect, the prebiotics are the short-chain fructo-oligosaccharides (forsimplicity shown herein below as FOSs-c.c); said FOSs-c.c. are notdigestible carbohydrates, generally obtained by the conversion of thebeet sugar and including a saccharose molecule to which three glucosemolecules are bonded.

The compositions of the invention may comprise pharmaceuticallyacceptable excipients or carriers. Examples of such suitable excipientsmay be found in the reference [103]. Acceptable carriers or diluents fortherapeutic use are well known in the pharmaceutical art and aredescribed, for example, in reference [104]. Examples of suitablecarriers include lactose, starch, glucose, methyl cellulose, magnesiumstearate, mannitol, sorbitol and the like. Examples of suitable diluentsinclude ethanol, glycerol and water. The choice of pharmaceuticalcarrier, excipient or diluent can be selected with regard to theintended route of administration and standard pharmaceutical practice.The pharmaceutical compositions may comprise as, or in addition to, thecarrier, excipient or diluent any suitable binder(s), lubricant(s),suspending agent(s), coating agent(s), solubilising agent(s). Examplesof suitable binders include starch, gelatin, natural sugars such asglucose, anhydrous lactose, free-flow lactose, beta-lactose, cornsweeteners, natural and synthetic gums, such as acacia, tragacanth orsodium alginate, carboxymethyl cellulose and polyethylene glycol.Examples of suitable lubricants include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Preservatives, stabilizers, dyes and even flavouring agentsmay be provided in the pharmaceutical composition. Examples ofpreservatives include sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid. Antioxidants and suspending agents may be alsoused.

The compositions of the invention may be formulated as a food product.For example, a food product may provide nutritional benefit in additionto the therapeutic effect of the invention, such as in a nutritionalsupplement. Similarly, a food product may be formulated to enhance thetaste of the composition of the invention or to make the compositionmore attractive to consume by being more similar to a common food item,rather than to a pharmaceutical composition. In certain embodiments, thecomposition of the invention is formulated as a milk-based product. Theterm “milk-based product” means any liquid or semi-solid milk- orwhey-based product having a varying fat content. The milk-based productcan be, e.g., cow's milk, goat's milk, sheep's milk, skimmed milk, wholemilk, milk recombined from powdered milk and whey without anyprocessing, or a processed product, such as yoghurt, curdled milk, curd,sour milk, sour whole milk, butter milk and other sour milk products.Another important group includes milk beverages, such as whey beverages,fermented milks, condensed milks, infant or baby milks; flavoured milks,ice cream; milk-containing food such as sweets.

In certain embodiments, the compositions of the invention contain asingle bacterial strain or species and do not contain any otherbacterial strains or species. Such compositions may comprise only deminimis or biologically irrelevant amounts of other bacterial strains orspecies. Such compositions may be a culture that is substantially freefrom other species of organism.

The compositions for use in accordance with the invention may or may notrequire marketing approval.

In some cases, the lyophilised bacterial strain is reconstituted priorto administration. In some cases, the reconstitution is by use of adiluent described herein.

The compositions of the invention can comprise pharmaceuticallyacceptable excipients, diluents or carriers.

In certain embodiments, the invention provides a pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in need thereof; and wherein the disorder iscancer, such as neuroblastoma, brain cancer, melanoma, prostate cancer,colorectal cancer, breast cancer, lung cancer, liver cancer or gastriccancer. In a further embodiment the cancer is ovarian cancer, cervicalcancer, glioblastoma, carcinoma, chronic lymphocyte leukemia, lymphomaor haematological malignancies.

In certain embodiments, the invention provides pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat or prevent adisease or condition mediated by MAP2. In preferred embodiments, saiddisease or condition is cancer, such as neuroblastoma, brain cancer,melanoma, prostate cancer, colorectal cancer, breast cancer, lungcancer, liver cancer or gastric cancer. In a further embodiment thecancer is ovarian cancer, cervical cancer, glioblastoma, carcinoma,chronic lymphocyte leukemia, lymphoma or haematological malignancies.

In certain embodiments, the invention provides pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat or prevent adisease or condition mediated by B3 Tubulin. In preferred embodiments,said disease or condition is cancer, such as neuroblastoma, braincancer, melanoma, prostate cancer, colorectal cancer, breast cancer,lung cancer, liver cancer or gastric cancer. In a further embodiment thecancer is ovarian cancer, cervical cancer, glioblastoma, carcinoma,chronic lymphocyte leukemia, lymphoma or haematological malignancies.

In certain embodiments, the invention provides pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat or prevent adisease or condition mediated by DRD2. In preferred embodiments, saiddisease or condition is cancer, such as neuroblastoma, brain cancer,melanoma, prostate cancer, colorectal cancer, breast cancer, lungcancer, liver cancer or gastric cancer. In a further embodiment thecancer is ovarian cancer, cervical cancer, glioblastoma, carcinoma,chronic lymphocyte leukemia, lymphoma or haematological malignancies.

In certain embodiments, the invention provides pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat or prevent adisease or condition mediated by HDAC. In preferred embodiments, saiddisease or condition is cancer, such as neuroblastoma, brain cancer,melanoma, prostate cancer, colorectal cancer, breast cancer, lungcancer, liver cancer or gastric cancer, ovarian cancer, cervical cancer,glioblastoma, carcinoma, chronic lymphocyte leukemia, lymphoma orhaematological malignancies.

In certain embodiments, the invention provides pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat or prevent adisease or condition mediated by pro-inflammatory cytokines, such asIL-1β, TNF-α, MIP-3α, IL-23 or IL-6. In a preferred embodiment, theinvention provides pharmaceutical composition comprising: a bacterialstrain of the invention; and a pharmaceutically acceptable excipient,carrier or diluent; wherein the bacterial strain is in an amountsufficient to treat or prevent a disease or condition mediated by TNF-α.In a preferred embodiment, the invention provides pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat or prevent adisease or condition mediated by IL-8. In a preferred embodiment, theinvention provides pharmaceutical composition comprising: a bacterialstrain of the invention; and a pharmaceutically acceptable excipient,carrier or diluent; wherein the bacterial strain is in an amountsufficient to treat or prevent a disease or condition mediated by CD11b.In preferred embodiments, said disease or condition is cancer, such asneuroblastoma, brain cancer, melanoma, prostate cancer, colorectalcancer, breast cancer, lung cancer, liver cancer or gastric cancer. In afurther embodiment said cancer is ovarian cancer, cervical cancer,glioblastoma, carcinoma, chronic lymphocyte leukemia, lymphoma orhaematological malignancies.

In certain embodiments, the invention provides pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat or prevent adisease or condition mediated by Casp3. In preferred embodiments, saiddisease or condition is cancer, such as neuroblastoma, brain cancer,melanoma, prostate cancer, colorectal cancer, breast cancer, lungcancer, liver cancer or gastric cancer. In a further embodiment saidcancer is ovarian cancer, cervical cancer, glioblastoma, carcinoma,chronic lymphocyte leukemia, lymphoma or haematological malignancies.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the amount of the bacterial strain is from about1×10³ to about 1×10¹¹ colony forming units per gram with respect to aweight of the composition.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the composition is administered at a dose of 1 g, 3g, 5 g or 10 g.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the composition is administered by a methodselected from the group consisting of oral, rectal, subcutaneous, nasal,buccal, and sublingual.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a carrier selected from the group consisting oflactose, starch, glucose, methyl cellulose, magnesium stearate, mannitoland sorbitol.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a diluent selected from the group consisting ofethanol, glycerol and water.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising an excipient selected from the group consistingof starch, gelatin, glucose, anhydrous lactose, free-flow lactose,beta-lactose, corn sweetener, acacia, tragacanth, sodium alginate,carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodiumstearate, magnesium stearate, sodium benzoate, sodium acetate and sodiumchloride.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, further comprising at least one of a preservative, anantioxidant and a stabilizer.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a preservative selected from the groupconsisting of sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein said bacterial strain is lyophilised.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein when the composition is stored in a sealedcontainer at about 4° C. or about 25° C. and the container is placed inan atmosphere having 50% relative humidity, at least 80% of thebacterial strain as measured in colony forming units, remains after aperiod of at least about: 1 month, 3 months, 6 months, 1 year, 1.5years, 2 years, 2.5 years or 3 years.

Culturing Methods

The bacterial strains for use in the present invention can be culturedusing standard microbiology techniques as detailed in, for example,references [105, 107].

The solid or liquid medium used for culture may be YCFA agar or YCFAmedium. YCFA medium may include (per 100 ml, approximate values):Casitone (1.0 g), yeast extract (0.25 g), NaHCO₃ (0.4 g), cysteine (0.1g), K₂HPO₄ (0.045 g), KH₂PO₄ (0.045 g), NaCl (0.09 g), (NH₄)₂SO₄ (0.09g), MgSO₄. 7H₂O (0.009 g), CaCl₂ (0.009 g), resazurin (0.1 mg), hemin (1mg), biotin (1 μg), cobalamin (1 μg), p-aminobenzoic acid (3 μg), folicacid (5 μg), and pyridoxamine (15 μg).

Bacterial Strains for Use in Vaccine Compositions

The inventors have identified that the bacterial strains of theinvention are useful for treating or preventing diseases or conditionsassociated with reduce immune activity. This is likely to be a result ofthe effect that the bacterial strains of the invention have on the hostimmune system. Therefore, the compositions of the invention may also beuseful for preventing diseases or conditions such as cancer, whenadministered as vaccine compositions. In certain such embodiments, thebacterial strains of the invention may be killed, inactivated orattenuated. In certain such embodiments, the compositions may comprise avaccine adjuvant. In certain embodiments, the compositions are foradministration via injection, such as via subcutaneous injection.

In certain embodiments, a bacterial strain of the species Megasphaeramassiliensis reduces the levels of formic acid. Formic acid is theconjugate base of formate which has been implicated in disruptingmitochondrial electron transport and energy production by inhibitingcytochrome oxidase activity, the terminal electron acceptor of theelectron transport chain. Accordingly, the reduction of formic acid, andthus formate, would reduce incidence of cell death via either cytochromeoxidase inhibition of accumulation of reactive oxygen species.Accordingly, in certain embodiments, a bacterial strain of the speciesMegasphaera massiliensis stimulates the immune system in the treatmentof disease by reducing the levels of formic acid.

General

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., references[108] and [109,115], etc.

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x is optional andmeans, for example, x±10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

References to a percentage sequence identity between two nucleotidesequences means that, when aligned, that percentage of nucleotides arethe same in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in section 7.7.18 of ref.[116]. A preferred alignment is determined by the Smith-Watermanhomology search algorithm using an affine gap search with a gap openpenalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. TheSmith-Waterman homology search algorithm is disclosed in ref. [117].

Unless specifically stated, a process or method comprising numeroussteps may comprise additional steps at the beginning or end of themethod, or may comprise additional intervening steps. Also, steps may becombined, omitted or performed in an alternative order, if appropriate.

Various embodiments of the invention are described herein. It will beappreciated that the features specified in each embodiment may becombined with other specified features, to provide further embodiments.In particular, embodiments highlighted herein as being suitable, typicalor preferred may be combined with each other (except when they aremutually exclusive).

MODES FOR CARRYING OUT THE INVENTION Example 1—MRx0029 Induces a MaturePhenotype in SH-SY5Y Cells

Introduction

The inventors sought to identify the effect of MRx0029 on the expressionof neurodifferentiation markers, β3Tubulin and MAP2 in neuroblastomacells. β3Tubulin is a marker of pre-differentiation in neurons and MAP2is a marker of mature (differentiated) neurons.

Bacterial Strain

Megasphaera massiliensis MRx0029

Cell Line

SH-SY5Y cells

Method

qPCR

SH-SY5Y were plated in 10 cm petri dishes a density of 2×10⁶ cells.After 24 h cells were treated in differentiation medium (growth mediumcontaining 1% FBS without RA) with 10% bacteria supernatants or YCFA+,10 uM RA, 200 uM hexanoic acid or 200 uM valproic acid, for 17 hrs.There after representative images were taken using phase contrast EVOSXL core microscope at 40×/0.65 magnification. Cells were collected, andtotal RNA was isolated according to RNeasy mini kit protocol (Qiagen).cDNAs were made using the high capacity cDNA reverse transcription kit(Applied Biosystems). Gene expression was measured using qPCR. GAPDH wasused as internal control. Fold change was calculated according to the2^((−ΔΔct)) method.

Immunolabeling and Cell Imaging

Cells were seeded onto 8-well chamber slides (Marienfeld LaboratoryGlassware) at 5×10⁴ cells/well overnight and were treated with 10%bacterial supernatant for 24 h. For differentiation, cells were treatedwith 10 nM RA for 5 days before treating with cell-free bacterialsupernatant for 24 h.

Afterwards, the cells were fixed with 4% paraformaldehyde in PBS for 20minutes at room temperature (RT). Fixed cells were washed with PBS, andpermeabilized with 1% Triton X-100 in PBS for 10 minutes. After washingwith PBS, the slides were incubated with blocking buffer (4% BSA/PBS)for 1 h at RT before adding anti-MAP2 antibody or β3-tubulin (sc-74421and sc-80005 respectively, Santa Cruz Biotechnology Inc) diluted in 1%BSA/PBS for 12 h at 4° C. They were then washed twice with PBS, followedby incubation with Alexa Flour 488 conjugated anti-mouse (MolecularProbes Inc) and Alexa Flour 594 conjugated Phalloidin (ab176757, Abcam)for 1 h at RT. After washing 3× with PBS, the slides were staining withDAPI and mounted with Vectashield® (Vector Laboratories). Slides wereviewed using a Axioskop 50 microscope (Zeiss) equipped with a 63×/1.2 WKorr objective and filter sets suitable for detection of thefluorochromes used. Manual exposure times for the digital acquisition ofimages immuno-labelled with MAP-2 were kept constant allowing comparisonbetween different wells and treatments. Phalloidin (F-actin) and DAPIexposure times varied to suit the field of view. Randomised fields ofview were acquired using a QImaging camera controlled by Image Pro Plussoftware. Images were saved as TIFF files and opened in Adobe PhotoshopCC 2015.1.2. Images of the MAP-2, DAPI and Phalloidin images were thenoverlaid and merged. Representative images were selected to illustratethe differences in abundance and location of the proteins examined.

Immunoblotting

SH-SY5Y cells cultured under the indicated conditions described above,treated with MRx0029 for 24 h and then lysed in RIPA buffer containingcocktail of protease inhibitors (Roche Diagnostics, UK). Proteinconcentration was estimated using the BCA protein assay kit (PierceBiotechnology, Rockford, Ill.), separated by SDS-PAGE and transferred toa PVDF membrane. Membranes were then blocked with 5% non-fat dry milk or5% BSA and incubated overnight at 4° C. with the primary antibodies(respectively MAP2 and β3-tubulin). The blots were then incubated withthe appropriate horseradish peroxidase (HRP)-conjugated secondaryantibody, and proteins were detected by chemiluminescence detection kit(Pierce Biotechnology, Rockford, Ill.). For both MAP2 and β3-tubulin,β-actin served as a control to monitor protein loading variabilityamongst samples.

Results

MRx0029 induces the expression of β3Tubulin in undifferentiatedneuroblastoma SH-SY5Y cells. FIG. 1 shows that treatment with MRx0029increases the expression of β3Tubulin in comparison to untreated SH-SY5Ycells.

MRx0029 induces the expression of MAP2 in undifferentiated neuroblastomaSH-SY5Y cells. FIGS. 2A-2C show that treatment with MRx0029significantly increased the expression of MAP2 in comparison tountreated SH-SY5Y cells.

Discussion

The results show that MRx0029 may be an effective composition inpromoting differentiation, in particular neuronal differentiation.Furthermore, the results show that MRx0029 may be an effectivecomposition in the treatment of brain cancer, in particularneuroblastoma, and melanoma, in particular metastatic melanoma.

Example 2—MRx0029 Decreases the Expression of DRD2 in SH-SY5Y Cells

Introduction

The inventors sought to identify the effect of MRx0029 on the expressionof DRD2 in neuroblastoma cells.

Bacterial Strain

Megasphaera massiliensis MRx0029

Cell Line

SH-SY5Y cells

Method

The inventors measured the change in DRD2 expression in neuroblastomacells using the same method as described in Example 1.

Results

MRx0029 decreases the expression of DRD2 in neuroblastoma SH-SY5Y cells.FIG. 3 shows that treatment with MRx0029 significantly decreased theexpression of DRD2 in comparison to untreated SH-SY5Y cells.

Discussion

This shows that MRx0029 may be an effective composition in the treatmentof cancer.

Example 3—MRx0029 Induces the Upregulation of Caspase 3 in SH-SY5Y Cells

Introduction

The inventors sought to identify the effect of MRx0029 on the expressionof Caspase 3 (Casp3) in neuroblastoma cells.

Bacterial Strain

Megasphaera massiliensis MRx0029

Cell Line

SH-SY5Y cells

Method

The inventors measured the change in Casp3 expression in neuroblastomacells.

Results

MRx0029 induces the upregulation of Casp3 in undifferentiatedneuroblastoma SH-SY5Y cells. FIG. 4 shows that treatment with MRx0029increases the expression of Casp3 in comparison to untreated SH-SY5Ycells. In particular, FIG. 4 shows that administration of MRx0029increased the expression of Casp3 threefold in comparison to thecontrol.

Discussion

In undifferentiated SH-SY5Y cells, the increase in Casp3 gene expressionby MRx0029 could be linked to both cell differentiation and theinduction of programmed cell death, such as apoptosis.

Caspase 3 is an executioner caspase and therefore is associated withapoptosis. Dysregulated apoptosis has been implicated in cancers andtherefore, the results show that MRx0029 may be an effective compositionin the treatment of cancer.

It has been shown that caspases have roles in cellular differentiation.Therefore the increase in Casp3 expression after treatment with MRx0029shows that MRx0029 may be an effective composition to increase celldifferentiation.

Example 4—MTT Assay in SH-SY5Y Cells

Introduction

The inventors sought to identify the effect of MRx0029 on neuroblastomacell viability, using the MTT assay, which is a widely used method forassessing the cell metabolic activity which reflects in the number ofviable cells.

Bacterial Strains

Megasphaera massiliensis MRx0029

Cell Line

SH-SY5Y cells

Method

MTT Assay

SH-SY5Y cells were plated at a seeding density of 10,000 cells/well, 24hrs later cells were treated in 100 ul of 1% FBS growth media withdifferent concentrations (express as percentage v/v) of cell-freebacterial supernatants from stationary phase cultures for 22 h.Thereafter 10 μl of MTT solution was added and cells were incubated inCO2 incubator for 4 h, at the end of this time 100 μl of isopropanolwith 0.04 HCL was added to each well. The absorbance was measured at 560nm wavelength and a reference wavelength of 655 nm. MTT assay kit waspurchased from Merck Millipore (Cat n. CT01).

Results

MRx0029 showed dose dependent effects on neuroblastoma cell viability,wherein 10% MRx0029 reduced viability by approximately 70% in comparisonto the control. Treatment with 10% of MRx0029 cell-free bacterialsupernatant showed a decrease in cell viability. The results of theexperiments are shown in FIG. 5 .

Discussion

This shows that MRx0029 may be an effective composition in increasingcell death, and therefore MRx0029 may be an effective composition foruse in the treatment of cancer.

Example 5—Basic Cell Phenotyping on PBMCs from Healthy Donors

Bacterial Strain

Megasphaera massiliensis MRx0029

Method

PBMCs Treatment

Frozen healthy human PBMCs were purchased from Stem Cells Technologies(Cambridge UK). Briefly cells were thaw and left to rest overnight infull growth media (RPMI 1640 with 10% FBS, 2 mM L. Glutamine, 55 μM2-Mercapethanol and 100 U/ml penicillin, 100 μg/ml streptomycin) in CO2incubator at 37° C. For the experiment cells were plated at a density of750,000 Cell/well in 48 well plates and treated in full growth mediawith 10% bacteria supernatants in the presence or absence of 1 ng/mlLPS. Cell culture media was added to untreated wells. Cells were left torest for 72 h, thereafter cell free supernatants were collected and spundown for 3 minutes at 10,000 g at 4° C. Samples were stored at −80° C.for cytokine analysis.

Immunophenotyping

1.5×10⁶ cells per sample were stained with viability fixable dye(Miltenyi) to discriminate between live and dead cells for 10 min at RT.Afterwards the cells were stained with the cocktail of antibodies listedbelow (Miltenyi) for basic immunophenotyping (CD3/CD4/CD8/CD25/CD127 andCD19) and incubated for 10 min at RT.

Experiments were carried out to measure the percentage of the followingcell populations:

-   -   CD4+CD3+ cells (markers of CD4 T-helper cells)    -   CD4+CD25+ cells (markers of CD4+ activated cells)    -   CD25++CD17− cells out of the CD4+ cell population (markers of        Tregs cells)    -   CD8+CD3+ cells (markers of cytotoxic T cells)    -   CD25+CD8+ cells (markers of CD8+ activated cells)    -   CD19+CD3− cells (markers of B cells).

The ratio of CD8+/Tregs and the ratio of activated CD8/Treg cells weredetermined.

Antibodies

Aria AB-Fluorochrome V2 CD3-VioBlue APC CD4-APC-Vio 770 Cy7 PE-Cy7CD8-PE-Vio 770 PE CD25-PE APC CD127-APC FITC CD19-VioBright 515

Results

The results of the experiments are shown in FIG. 6 .

The most surprising result is the effect of MRx0029 treatment on thepercentage of CD25++CD17− cells, which represent Treg cells (see FIG.6C). MRx0029 selectively reduced the percentage of Tregs in the PBMCpopulation. MRx0029 treatment did not significantly change thepercentage of CD4 T-helper cells, CD4+ activated cells, cytotoxic Tcells, CD8+ activated cells or B cells.

Treatment with MRx0029 increased the ratio of CD8+/Tregs and the ratioof activated CD8/Treg cells in comparison to untreated cells (see FIG.6G and FIG. 6H).

Discussion

The observation that treatment with MRx0029 selectively decreased thepercentage of Tregs, thereby increasing the ratios of CD8/Treg andactivated CD8/Treg is surprising because MRx0029 produces butyrate, andbutyrate production has been associated with increases in the populationof Tregs.

Basic immunophenotyping profile of MRx0029 in PBMCs from healthy donorssuggests that treatment with MRx0029 decreases the relative percentageof Tregs in the lymphocyte population, which reflects in an increasingratio between CD8/Tregs cells.

This shows that MRx0029 may be an effective composition in stimulatingthe immune response, and decreasing immune suppression by Tregs. Theresults also show that MRx0029 may be an effective composition for usein the treatment of cancer.

Example 6—Cytokine Analysis of PBMCs from Healthy Donors

Introduction

The inventors sought to further analyse PBMCs post-incubation withMRx0029. The inventors analysed the expression of particular cytokinesfrom PBMCs upon treatment with MRx0029, including pro-inflammatorycytokines TNF-α, IL-1β and IL-23.

Bacterial Strain

Megasphaera massiliensis MRx0029

Method

PBMCs Treatment

PBMCs were treated as described in Example 5.

Cytokine Quantification

Cytokine quantification was conducted using a ProcartaPlex multipleximmunoassay following the manufacturer's recommendations (Thermo FischerScientific). Briefly, 50 μl of cell-free co-culture supernatants wereused for cytokine quantification using a MAGPIX® MILLIPLEX® system(Merck) with the xPONENT software (Luminex, Austin, Tex., USA). Data wasanalysed using the MILLIPLEX® analyst software (Merck) using a5-parameter logistic curve and background subtraction to convert meanfluorescence intensity to pg/ml values.

Results

The results for the Cytokine analysis of MRx0029 in PBMC culture fromhealthy donors showed an immune-stimulatory signature for MRx0029. Inparticular, MRx0029 treatment increased the expression of TNF-α, IL-1βand IL-23.

The results also show that treatment with MRx0029 increased theexpression of MIP-3α, IL-6 and IL-10. The expression levels of MCP-1,CXCL9, and GMCSF were similar to controls.

Discussion

This shows that MRx0029 has immunostimulatory properties, and may be aneffective composition for immunostimulation. The results also show thatMRx0029 may be an effective composition in the treatment of cancer.

Example 7—Flow Cytometry Analysis of Different Cell Populations

Introduction

The inventors sought to further analyse PBMCs post-incubation withMRx0029. Flow cytometry analysis was used to determine the percentagesof CD4 cells (CD4+CD3+), Tregs (CD25++CD127−) CD8 cells (CD8+CD3+) and Bcells (CD19+CD3−).

Method

PBMC Treatment

PBMCs were treated as described in Example 5.

Flow Cytometry Analysis of Different Cell Populations

1.5×10⁶ cells per sample were stained with viability fixable dye(Miltenyi) to discriminate between live and dead cells for 10 min at RT.Afterwards the cells were stained with the cocktail of antibodies listedbelow (Miltenyi) for basic immunophenotyping (CD3/CD4/CD8/CD25/CD127 andCD19) and incubated for 10 min at RT. Cells were then washed andresuspended in PBS and immediately analysed using a FACS Aria IIequipped with Blue (488 nm), Red (633 nm) and Violet (405 nm) lasers.FMOs were included throughout all the experiments. For the analysisFlowjo version 10.4.2 software (FlowJo, LLC) was used.

Results

The results for the flow cytometry experiments are shown in FIGS. 8A-8C.

FIG. 8A shows that 0.73% of PBMCs were Tregs (CD25++CD127−).

Discussion

The results show that MRx0029 may be useful for downregulating Tregs.Furthermore, the results show that MRx0029 may be an effectivecomposition in stimulating the immune response, and decreasing immunesuppression by Tregs. The results also show that MRx0029 may be aneffective composition for use in the treatment of cancer.

Example 8—MRx0029 Increases IL-8 Secretion in MG U373 Cells

Introduction

The inventors sought to identify the effect of MRx0029 on the secretionof IL-8 in neuroblastoma cells. Human glioblastoma astrocytoma cellswere treated with compositions comprising MRx0029 in combination withLPS to observe their ability to modulate the levels of IL-8. IL-8 is apro-inflammatory cytokine secreted predominantly by macrophages withimmune-stimulatory effects.

Bacterial Strain

Megasphaera massiliensis MRx0029

Cell Line

MG U373 is a human glioblastoma astrocytoma derived from a malignanttumour and were purchased from Sigma-Aldrich (cat n. 08061901-1VL). MGU373 human glioblastoma astrocytoma cells were grown in MEM (SigmaAldrich, cat n. M-2279) supplemented with 10% FBS, 1% Pen Strep, 4 mML-Glut, 1×MEM Non-essential Amino Acid solution and 1× Sodium Piruvate.

Method

Once grown the MG U373 cells were plated on 24-well plate at 100,000cells/well. The cells were treated with LPS (1 ug/mL) alone or with 10%of bacteria supernatant from MRx0029 for 24 h. LPS is a known stimulatorof proinflammatory cytokines such as IL-8. Afterwards the cell freesupernatants were collected, centrifuged at 10,000 g for 3 min at 4° C.IL-8 was measured using Human IL-8 ELISA Kit from Peprotech (cat n.#900-K18) according to manufacturer instruction.

Results

The results of these experiments are shown in FIG. 9 . Treatment thecells with the bacteria strains lead to an increase in IL-8 secretionindependently of the presence of LPS.

Discussion

The results show that MRx0029 may be useful for increasing IL-8secretion. Therefore, compositions of the invention may be useful in thetreatment of disease, in particular diseases characterised by reducedimmune activation and diseases treatable by an increased immuneresponse.

Example 9—MRx0029 Reduces the Levels of Histone Deacetylase Activity inHT-29 Cells

Introduction

The ability of compositions comprising MRx0029 to alter histonedeacetylase activity was investigated. HDACi have been shown to causegrowth arrest, differentiation, apoptosis, reduction of angiogenesis andmodulation of the immune response in a variety of cancer cell lines.

Bacterial Strain

Megasphaera massiliensis MRx0029

Cell Line

The cell line HT-29 was used because histone deacetylase is present.

Method

Cell free supernatants of stationary phase bacterial cultures wereisolated by centrifugation and filtering in a 0.22 uM filter. HT-29cells were used 3 days post confluence and stepped down in 1 mL DTS 24hours prior to commencement of the experiment. The HT-29 cells werechallenged with 10% cell free supernatant diluted in DTS and was is leftto incubate for 48 hours. Nuclease proteins were then extracted usingthe Sigma Aldrich Nuclease extraction kit and samples were snap frozenprior to HDAC activity measurement. HDAC activity was assessedfluorometrically using the Sigma Aldrich (UK) kit.

Results

The results of the experiments are shown in FIG. 10 . FIG. 109 showsthat MRx0029 is able reduce the levels of histone deacetylase activity.

Discussion

The results show that MRx0029 is a promising candidate for use intreating or preventing diseases characterised by epigenetic aberrations,through inhibition of HDAC activity. Cancer is a disease characterisedby epigenetic aberrations. Furthermore, HDAC inhibitors (HDACi) are anemerging class of promising anti-cancer drugs that have been shown tocause growth arrest, differentiation, apoptosis, reduction ofangiogenesis and modulation of the immune response in a variety ofcancer cell lines. Therefore, the results shows that compositions of theinvention may be effective for use in the treatment or prevention ofcancer.

Example 10—Further Analysis of the Mechanism of Histone DeacetylationInhibition

Introduction

The gut microbiota, with its immense diversity and metabolic capacity,represents a huge metabolic reservoir for production of a vast varietyof molecules with potential to influence HDAC activity. The inventorstherefore sought to determine which metabolites are responsible for HDACinhibition and further elucidate the mechanisms by which inhibition isachieved.

Bacterial Strain

Megasphaera massiliensis MRx0029

Method

Bacterial Culture and Cell-Free Supernatant Collection

Pure cultures of bacteria were grown anaerobically in YCFA+ broth untilthey reached their stationary growth phase. Cultures were centrifuged at5,000×g for 5 minutes and the cell-free supernatant (CFS) was filteredusing a 0.2 μM filter (Millipore, UK). 1 mL aliquots of the CFS werestored at −80° C. until use. Sodium butyrate, hexanoic and valeric acidwere obtained from Sigma Aldrich (UK) and suspensions were prepared inYCFA+ broth.

SCFA and MCFA Quantification of Bacterial Supernatants Short chain fattyacids (SCFAs) and medium chain fatty acids (MCFAs) from bacterialsupernatants were analysed and quantified by MS Omics APS as follows.Samples were acidified using hydrochloride acid, and deuterium labelledinternal standards where added. All samples were analyzed in arandomized order. Analysis was performed using a high polarity column(Zebron™ ZB-FFAP, GC Cap. Column 30 m×0.25 mm×0.25 μm) installed in a GC(7890B, Agilent) coupled with a quadropole detector (59977B, Agilent).The system was controlled by ChemStation (Agilent). Raw data wasconverted to netCDF format using Chemstation (Agilent), before the datawas imported and processed in Matlab R2014b (Mathworks, Inc.) using thePARADISe software described in [118].

Specific HDAC Activity Analysis

Specific HDAC inhibition activity was analysed for HDAC1, 2, 3, 4, 5, 6,9 using fluorogenic assay kits for each type of HDAC (BPS Bioscience,CA). Assays were conducted according to manufacturer's instructions andeach sample were performed in replicates. Cell free supernatants werediluted 1 in 10 and exposed to specific HDAC proteins provided in thekit to maintain consistency between methods.

Results

Histone Deacetylase-Inhibiting Gut Commensal Microbial Metabolites areButyrate and Valeric Acid

MRx0029, whose supernatant showed strong HDAC inhibition in both HT29whole cells and HT29 cell lysates (see FIG. 11A), produced valeric acidand hexanoic acid at mean concentrations of 5.08 mM and 1.60 mM,respectively (see FIG. 11B).

To investigate which metabolites were responsible for the strain-inducedHDAC inhibition, different concentrations of hexanoic acid, valeric acidand sodium butyrate were measured for their HDAC inhibition on wholeHT-29 cells and on HT-29 cell lysate. The results in FIG. 11C showsignificant (P<0.05) inhibition of HDAC activity by sodium butyrate onwhole cells as well as on the cell lysate, while hexanoic acid did notshow significant inhibitory activity. Valeric acid also inhibited HDACactivity on whole cells as well as on the cell lysate (*(p<0.05),**(p<0.005), ***(P<0.001), *(p<0.0001)).

Potent Total HDAC Inhibitors Investigated Target Class I HDACs.

The specific HDAC inhibition profile of the test bacteria strain wasinvestigated. Specific HDAC inhibition assays (BPS Bioscience, CA) werecarried out for Class I and Class II HDACs. The ability of the bacterialstrain to inhibit HDAC enzymes was compared to butyrate, hexanoic andvaleric acid. Our results demonstrate that MRx0029 is a very potentinhibitor of Class 1 HDAC enzymes (HDAC1, 2 and 3). Inhibition of classII HDACs was not as significant (data not shown).

Discussion

The strain with HDAC inhibitory activity produced significant amounts ofvaleric acid and hexanoic acid as well as significant amounts of sodiumbutyrate (FIG. 11B). When tested as pure substances, valeric acid andsodium butyrate resulted in significant HDAC inhibition (p<0.0001).

Interestingly, the results for specific HDAC activity show that thetested strain is a potent inhibitor of Class I HDACs, and particularlyHDAC2 (FIG. 12 and FIG. 13 ). Class I HDACs (HDAC1, 2, 3 and 8) residein the nucleus and are ubiquitously expressed in several human celltypes. HDACs 1-3 share more than 50% homology, but have distinctstructures and cellular functions [119]. They are primarily involved incell survival, proliferation and differentiation, and thus theirinhibition may be useful is wide array of diseases [120]; [121]; [122];[123]; [124]. Therefore, compositions of the invention may beparticularly useful for treatment of diseases where Class I HDACsactivity is upregulated. In particular, compositions of the inventionmay be particularly useful for treatment of cancers where Class I HDACsactivity is upregulated. For example, compositions of the invention maybe particularly useful for treatment of cancers where HDAC2 activity isupregulated.

Example 11—Modulation of Gut Barrier Function and Gut Permeability byMRx0029

Introduction

The ability of MRx0029 to cause any intestinal barrier dysfunction wasinvestigated. HT29-mtx epithelial, mucin-producing cell monolayers [125]were used as an in vitro model to evaluate gut barrier disruption andimmune stimulation following treatment with MRx0029.

Bacterial Strain

Megasphaera massiliensis MRx0029

Methods

RNA Extraction and qPCR Analysis

Total RNA was extracted using the RNeasy mini kit (Qiagen, Manchester,JUK) according to the manufacturer's instructions, and the RNAconcentration determined by absorbance at 260/280 nm using aspectrophotometer (nano-Drop ND-1000; Thermo Scientific, Wilmington,Del.). For mRNA expression analysis, cDNA was prepared from total RNAusing the High-Capacity cDNA reverse transcription kit (AppliedBiosystems, UK) according to the manufacturer's instructions. Thereverse transcription reactions were performed in a Thermo cycler(Biometra, Germany) at 25° C. for 10 min, 37° C. for 120 min, and 85° C.for 5 min, hold on at 4° C. Resulting cDNA was amplified in duplicatesby the SYBR-Green PCR assay, and products were detected on QuantStudio 6flex real-time PCR machine (Applied Biosystems, UK) using a standardisedprofile (initial denaturation of 95° C. for 10 minutes, followed by 40cycles of 15 seconds of denaturation at 95° C. and 60 seconds ofannealing/extension at 60/65° C., depending on the primers. Adissociation stage was added after the 40 cycles to generate a meltingcurve. Analysis was performed using the Applied Biosystems QuantStudioReal-Time PCR Software v1.2.

Results

Differentiated HT29-mtx cells exposed to phorbol 12-myristate-13-acetate(PMA) secreted a significant amount of IL-8; in contrast treatment for24 h with MRx0029 bacterial supernatants, induced an even lowersecretion of IL-8 compared than both untreated and YCFA+-treated cells(FIG. 14A).

The ability of MRx0029 to regulate epithelial permeability by modifyingintracellular signal transduction involved in the expression andlocalization of proteins involved in the gut barrier formation was theninvestigated.

RNA was isolated and Quantitative RT-PCR (qRT-PCR) analysis wasperformed to characterize the changes in gene expression of tightjunction proteins during incubation with MRx0029. The administration ofMRx0029 enhanced Occludin, Villin, Tight Junction Protein 1 and 2(respectively TJP1 and TJP2) mRNA expression after 2 h incubation (FIG.14B).

The in vitro results were compared with data from the ex vivo parallelanalysis on the gut of mice fed with MRx0029. Gene expression of TJP1and Occludin was quantified in the colon and ileum. The ex vivo dataperfectly mirror the in vitro data as MRx0029 was able to significantlyup-regulate TJP1 and Occludin (p=0.073) in the colon region of themurine intestine (FIG. 14C+14D). MRx0029 was also able to decrease thepermeability function in the colon of the same mice (FIG. 14F).

Discussion

The results show that MRx0029 is able to regulate epithelialpermeability by modifying intracellular signal transduction involved inthe expression and localization of proteins involved in gut barrierfunction (e.g. Occludin, Villin, TJP1 and TJP2). The results thereforeshow that MRx0029 functions to increase gut barrier function and reducegut permeability. Therefore, compositions of the invention are effectivefor the treatment or prevention of diseases or conditions that arecharacterised by reduction of gut barrier function or increased gutpermeability.

Example 12

Introduction

The inventors sought to analyse expression of genes for inflammatorymarkers in brain tissue from the hippocampus, amygdala and prefrontalcortex of mice fed with MRx0029. The inventors also explored the effectson cytokine production from the spleen in the same mice administeredMRx0029.

Bacterial Strain

Megasphaera massiliensis MRx0029

Methods

Animals

BALBc (Envigo, UK) adult male mice were group housed under a 12 hlight-dark cycle; standard rodent chow and water were available adlibitum. All experiments were performed in accordance with Europeanguidelines following approval by University College Cork Animal EthicsExperimentation Committee. Animals were 8 weeks old at the start of theexperiment.

Study Design

Animals were allowed to habituate to their holding room for one weekafter arrival into the animal unit. They receive oral gavage (200 μLdose) of live biotherapeutics at a dose of 1×10⁹ CFU for 6 consecutivedays between 15:00 and 17:00. On day 7, the animals are decapitated, andtissues are harvested for experimentation.

Tissue Collection

Animals were sacrificed in a random fashion regarding treatment andtesting condition; sampling occurred between 9:00 a.m. and 2:30 p.m.Trunk blood was collected in potassium EDTA (Ethylene Diamine TetraAcetic Acid) tubes and spun for 15 min at 4000 g. Plasma was isolatedand stored at −80° C. for further analysis. The brain was quicklyexcised, dissected and each brain region was snap-frozen on dry ice andstored at −80° C. for further analysis. Spleen was removed, collected in5 mL RPMI media (with L-glutamine and sodium bicarbonate, R8758Sigma+10% FBS (F7524, Sigma)+1% Pen/Strep (P4333, Sigma)) and processedimmediately after culls for ex-vivo immune stimulation. Intestinaltissue (23 cm segments of ileum and colon closest to the caecum wereexcised, and the furthest 1 cm 2 cm of tissue from the caecum were used)were mounted into the Ussing chambers for intestinal permeability assay.The caecum was removed, weighted and stored at −80° C. for SCFAsanalysis.

Spleen Cytokine Assay

Spleens were collected immediately in 5 mL RPMI media followingsacrifice and cultured immediately. Spleen cells were first homogenisedin this RPMI media, followed by 5 mins incubation with 1 ml of RBC lysisbuffer (11814389001 ROCHE, Sigma). A further 10 ml of RPMI media wasadded, followed by 200 G centrifugation for 5 mins. The supernatant wasthen filtered through 40 um strainer. Cells were counted and seeded(4,000,000/mL media). After 2.5 h of adaptation, cells were stimulatedwith lipopolysaccharide (LPS-2 μg/ml) or concanavalin A (ConA-2.5 μg/ml)for 24 h. Following stimulation, the supernatants were harvested toassess the cytokine release using Proinflammatory Panel 1 (mouse) V-PLEXKit (Meso Scale Discovery, Maryland, USA) for TNF-α, IL-10, IL-1β,Interferon γ, CXCL2 and IL6. The analyses were performed using MESOQuickPlex SQ 120, SECTOR Imager 2400, SECTOR Imager 6000, SECTOR S 600.

Gene Expression Analysis

Total RNA was extracted using the mirVana™ miRNA Isolation kit(Ambion/Llife technologies, Paisley, UK) and DNase treated (TurboDNA-free, Ambion/life technologies) according to the manufacturersrecommendations. RNA was quantified using NanoDrop™ spectrophotometer(Thermo Fisher Scientific Inc., Wilmington, Del., USA) according to themanufacturer's instructions. RNA quality was assessed using the AgilentBioanalyzer (Agilent, Stockport, UK) according to the manufacturer'sprocedure and an RNA integrity number (RIN) was calculated. RNA with RINvalue >7 was used for subsequent experiments. RNA was reversetranscribed to cDNA using the Applied Biosystems High Capacity cDNA kit(Applied Biosystems, Warrington, UK) according to manufacturer'sinstructions. Briefly, Multiscribe Reverse Transcriptase (50 U/μL)(1)(2)(1)(10) was added as part of RT master mix, incubated for 25° C.for 10 min, 37° C. for 2 h, 85° C. for 5 min and stored at 4° C.Quantitative PCR was carried out using probes (6 carboxyfluorescein—FAM) designed by Applied Biosystems to mouse specifictargeted genes, while using β-actin as an endogenous control.Amplification reactions contained 1 μl cDNA, 5 μl of the 2×PCR Mastermix (Roche), 900 nM of each primer and were brought to a total of 10 μlby the addition of RNase-free water. All reactions were performed intriplicate using 96-well plates on the LightCycler®480 System. Thermalcycling conditions were as recommended by the manufacturer (Roche) for55 cycles. To check for amplicon contamination, each run contained notemplate controls in triplicate for each probe used. Cycle threshold(Ct) values were recorded. Data was normalized using β-actin andtransformed using the 2-ΔΔCT method and presented as a fold change vs.control group.

Statistical Analysis

Normally distributed data are presented as mean±SEM; Non-parametricdatasets are presented as median with inter-quartile range. Unpairedtwo-tailed t-test were applied to analyse parametric data andMann-Whitney test was used for non-parametric. Spearman's rankcorrelation coefficient was employed for the correlation analysis in thepooled datasets. A p value <0.05 was deemed significant in all cases.

Results—Gene Expression

Expression of genes for inflammatory markers [IL-1β, IL6, CD11b, TNF-αand TLR-4] were analysed in brain tissue from the hippocampus, amygdalaand prefrontal cortex. FIGS. 15-25 show the changes in gene expressionafter MRx0029 treatment in the hippocampal, amygdala and prefrontalcortex. Treatment with MRx0029 significantly increased the expression ofTLR-4 in the amygdala (FIG. 20 ). Treatment with MRx0029 also increasedexpression of CD11b in the amygdala (FIG. 21 ).

Results—Effect on Cytokine Expression from Splenocytes

The ex-vivo splenocyte assay involves challenging the splenocytes (cellsisolated from the spleen—a main organ involved in immune defence), witha bacterio- or viral-mimetic challenge.

Treatment with MRx0029 led to a reduction in interferon-γ,interleukin-1β and interleukin-6 following a challenge with LPS (FIGS.26, 27 and 28 , respectively).

Treatment with MRx0029 led to an increase in the levels of thechemoattractant CXCL1 (FIG. 30 ).

Discussion

Treatment with MRx0029 significantly increased the expression of thepro-inflammatory cytokines TLR-4 and CD11b in the amygdala. Therefore,compositions of the invention may be useful in the treatment of disease,in particular diseases characterised by reduced immune activation anddiseases treatable by an increased immune response.

Example 13—Stability Testing

A composition described herein containing at least one bacterial straindescribed herein is stored in a sealed container at 25° C. or 4° C. andthe container is placed in an atmosphere having 30%, 40%, 50%, 60%, 70%,75%, 80%, 90% or 95% relative humidity. After 1 month, 2 months, 3months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years, atleast 50%, 60%, 70%, 80% or 90% of the bacterial strain shall remain asmeasured in colony forming units determined by standard protocols.

Example 14—Analysis of the Effect of M. massiliensis on the ERKSignalling Pathway

Materials and Methods

RNA Extraction and MAP2 qPCR Analysis

Cells were plated in 12-well plates at density of 2×10⁵ cells/well.After 24 h cells were treated with either DMSO or Vemurafenib (662005;EMD Millipore; VEMU; SKMEL28, SKMEL31, 451Lu, HT29 (1 μM) SKMEL2 (10 μM)or Azacytidine-C (A3656; Sigma Aldrich; AzaC; 5 μg/ml) or both drugs(VEMU+Aza) together, in presence of 10% bacteria supernatants or absenceof it (YCFA+). Total RNA was extracted using the RNeasy mini kit(Qiagen, Manchester, UK) according to the manufacturer's instructions,and the RNA concentration was determined by spectrophotometer at 260/280nm (NanoDrop ND-1000; Thermo Fisher Scientific, Loughborough). For mRNAexpression analysis, cDNA was prepared from 2000 ng of total RNA usingthe High-Capacity cDNA reverse transcription kit (Thermo Fisher,Loughborough) according to the manufacturer's instructions. The reversetranscription reactions were performed in a thermocycler (Biometra,Germany) at 25° C. for 10 min, 37° C. for 120 min, and 85° C. for 5 min.Resulting cDNA was amplified in duplicates by the SYBR-Green PCR assay,and products were detected on QuantStudio 6 flex real-time PCR machine(Applied Biosystems, UK) using a standardised profile (initialdenaturation of 95° C. for 10 minutes, followed by 40 cycles of 10seconds of denaturation at 95° C. and 30 seconds of annealing/extensionat 65° C.). A dissociation stage was added after the 40 cycles togenerate a melting curve. Analysis was performed using the AppliedBiosystems QuantStudio Real-Time PCR Software v1.2. The primer sequencesfor GAPDH, and MAP2 are shown below.

Western Blot Analysis

Following 24 h treatment with the appropriate drugs either in presenceof 10% bacterial supernatant or absence (YCFA+), protein extracts wereobtained by lysing cells in RIPA buffer (R0278; Sigma Aldrich)supplemented with protease inhibitors (cOmplete Protease InhibitorCocktail Tablets; Roche, Switzerland) and 1 mM/L sodium orthovanadate,0.5 mM/L PMSF. Protein quantification was done by the BCA protein assay.Equal amounts of total protein (20 μg/lane) were then separated bySDS-PAGE on 4-15% gradient gel (BioRad) and transferred topolyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific,Loughborough). After blocking with 5% BSA or nonfat dry milk in TBST (10mM Tris, pH 7.5, 150 mM NaCl, 0.5% Tween 20) for 60 min, membranes wereprobed with primary antibodies against phospho-ERK (9101S, 1:1000, CellSignalling; New England Biolabs (UK)) or total ERK (4696S, 1:1000, Cellsignalling; New England Biolabs (UK)).

Proteins of interest were detected with the appropriate HRP-conjugatedsecondary antibody (1:10,000, Thermo Fisher Scientific, Loughborough),developed with the ECL Western blotting Super Signal PicoPlus substrate(34577; Thermo Fisher Scientific, Loughborough), and visualised inChemidoc XRS Imager (BioRad).

Anchorage-Independent Growth (Soft Agar Growth Assay) in 96-Well Plates

A mixture of 25 μL prewarmed (37° C.) 2×appropriate growth medium (EMEMfor melanoma cell lines; DMEM high glucose for HT29) containing 20% FBS,4 mM L-Glu, 2×NEAA, 0.6% sodium bicarbonate, 200 U/mLpenicillin/streptomycin (Invitrogen), and 25 μL prewarmed (47° C.) 1.2%Noble Agar (A5431; Sigma Aldrich) were plated onto each well of a96-well microplate to serve as a prelayer for the assay. Ten microlitersof cell suspensions containing 0-2×10³ cells were mixed with 25 μL 2×growth media and 35 μL 0.8% Noble Agar in a 96-well round-bottompolypropylene microplate and transferred to the 96-well microplatecontaining the solidified prelayers. The cells were allowed to grow for2 days and then fed with media containing drugs in presence of 10%bacterial supernatants or YCFA+ every three days. They left to grow inthe humidified 37° C. incubator with 5% CO₂ for 1-2 weeks before softagar growth was scored using the CytoSelect 96 well cell transformationassay (CBA-130; Cell Biolabs) according to the manufacturer's protocol.Cell growth was measured using a Tecan Infinite F200 Pro SeriesMulti-Well Plate Reader (Tecan Biosystems), with excitation at 485 nmand emission at 530 nm.

Anchorage-Independent Growth (Soft Agar Growth Assay) in a 32-Mm Plate

A mixture of 1 mL of prewarmed (37° C.) 2×appropriate growth media (EMEMfor melanoma cell lines; DMEM high glucose for HT29) and 1 mL prewarmed(47° C.) 0.8% Noble Agar per plate (0.4% final agar) were mixed with 1mL cell suspension and seeded over a 0.6% agar/cell growth prelayer (2mL) in a 6-well plate. The cells were allowed to grow in the humidified37° C. incubator with 5% CO₂ for 21-28 days. They fed with drugs inabsence (YCFA+) or presence of 10% bacterial supernatant every threedays. Colonies were photographed using Evos XL Core microscope (ThermoFisher Scientific, Loughborough).

Clonogenic Assay

Cells were trypsinized and 200 cells/well were seeded in 12-well plates.After 48 h cells were treated with the appropriate drugs in absence(YCFA+) or presence of 10% bacterial supernatant and were re-fed everythree days. On day 21 after seeding, cells were fixed in ice-coldmethanol and stained with Crystal Violet blue. Colonies (0.50 cells)were counted and survival fraction was calculated as number of coloniesdivided by the number of plated cells (plating efficiency) of treateddivided by the plating efficiency of control.

Example 14A—SKMEL2 Melanoma Cell Line

The effects of the following treatments were assessed on the SKMEL2melanoma cell line (WT BRAF; N61R oncogenic mutation in Nras): (1)MRX0029; (2) Vemurafenib (VEMU) in YFCA+ medium; (3) VEMU and MRX029;(4) Azacytidine-C (Aza-c) in YFCA+ medium; (5) Aza-c and MRX029; (6)VEMU, Aza-c and MRX0029.

MAP2 gene expression in the SKMEL2 cell line was assessed using theprotocol in Materials and Methods, and the results are shown in FIG. 31. All treatments with MRX029 (alone or in combination with VEMU and/orAza-c) increased MAP2 gene expression relative to both negative controls(cell line only, and YCFA+). Clonogenic survival of the SKMEL2 cell linewas assessed using the protocol in Materials and Methods, and theresults are shown in FIG. 32 . Soft agar growth of the SKMEL2 cell linewas assessed using the protocol in Materials and Methods, and theresults are shown in FIG. 33 . VEMU+Aza-c improved soft agar growthinhibition by MRX029. ERK signalling in the SKMEL2 cell line wasassessed using the protocol in Materials and Methods, and the resultsare shown in FIG. 34 (VEMU, Aza-c and MRX029 was not assessed).

These results indicate that MRX0029 alone or in combination withVemurafenib and/or Azacytidine-C may have the effects of inducing MAP2gene expression in a melanoma cell line (SKMEL2). Furthermore,Vemurafenib+Azacytidine-C enhanced soft agar growth inhibition byMRX0029. On this basis, compositions of the invention are expected to beuseful in the treatment or prevention of various cancers, in particularmetastatic cancers, in particular metastatic melanoma.

Example 14B—SKMEL28 Melanoma Cell Line

The effects of the following treatments were assessed on the SKMEL28melanoma cell line (V600E oncogenic mutation in BRAF): (1) MRx0029; (2)Vemurafenib (VEMU) in YCFA+ medium; (3) VEMU and MRx0029; (4)Azacytidine-C (Aza-c) in YCFA+ medium; (5) Aza-c and MRX0029; (6) VEMU,Aza-c and MRx0029.

MAP2 gene expression in the SKMEL28 cell line was assessed using theprotocol in Materials and Methods, and the results are shown in FIG. 35. Clonogenic survival of the SKMEL28 cell line was assessed using theprotocol in Materials and Methods, and the results are shown in FIG. 36. MRx0029 in combination with VEMU and/or Aza-c decreased clonogenicsurvival relative to both negative controls (YCFA+ and cell line only).Soft agar growth of the SKMEL28 cell line was assessed using theprotocol in Materials and Methods, and the results are shown in FIG. 37. ERK signalling in the SKMEL28 cell line was assessed using theprotocol in Materials and Methods, and the results are shown in FIG. 38(VEMU, Aza-c and MRx0029 was not assessed). All treatments with MRx0029(alone or in combination with VEMU or Aza-c) reduced ERK signallingrelative to the negative control (YFCA+).

These results indicate that MRx0029 alone or in combination withVemurafenib and/or Azacytidine-C may have the effects of inhibiting ERKsignalling and decreasing clonogenic survival of a melanoma cell linecomprising the BRAF V600E mutation (SKMEL28). On this basis,compositions of the invention are expected to be useful in the treatmentor prevention of cancers, in particular those comprising oncogenic ERKsignalling, especially melanomas. In particular, compositions of theinvention are expected to be useful in the treatment or prevention ofsuch cancers comprising an oncogenic mutation in BRAF, in particular atposition 600, and especially the mutation BRAF V600E.

Example 14C—SKMEL31 Melanoma Cell Line

The effects of the following treatments were assessed on the SKMEL31melanoma cell line (heterozygous for BRAF V600E): (1) MRx0029; (2)Vemurafenib (VEMU) in YFCA+ medium; (3) VEMU and MRx0029; (4)Azacytidine-C (Aza-c) in YFCA+ medium; (5) Aza-c and MRx0029; (6) VEMU,Aza-c and MRx0029.

MAP2 gene expression in the SKMEL31 cell line was assessed using theprotocol in Materials and Methods, and the results are shown in FIG. 39. Clonogenic survival of the SKMEL31 cell line was assessed using theprotocol in Materials and Methods, and the results are shown in FIG. 40. Soft agar growth of the SKMEL31 cell line was assessed using theprotocol in Materials and Methods, and the results are shown in FIG. 41. VEMU, Aza-c and VEMU+Aza-c improved soft agar growth and clonogenicsurvival inhibition by MRx0029. ERK signalling in the SKMEL31 cell linewas assessed using the protocol in Materials and Methods, and theresults are shown in FIG. 42 (VEMU, Aza-c and MRx0029 in combination wasnot assessed). All treatments with MRx0029 (alone or in combination withVEMU or Aza-c) reduced ERK signalling relative to the negative control(YFCA+).

Example 14D—451Lu Melanoma Cell Line

The effects of the following treatments were assessed on the 451Lumelanoma cell line (V600E oncogenic mutation in BRAF): (1) MRx0029; (2)Vemurafenib (VEMU) in YFCA+ medium; (3) VEMU and MRx0029; (4)Azacytidine-C (Aza-c) in YFCA+ medium; (5) Aza-c and MRx0029; (6) VEMU,Aza-c and MRx0029.

MAP2 gene expression in the 451Lu cell line was assessed using theprotocol in Materials and Methods, and the results are shown in FIG. 43. All treatments with MRx0029 (alone or in combination with VEMU and/orAza-c) increased MAP2 gene expression relative to the cell line onlynegative control. Clonogenic survival of the 451Lu cell line wasassessed using the protocol in Materials and Methods, and the resultsare shown in FIG. 44 . All treatments with MRx0029 (alone or incombination with VEMU and/or Aza-c) decreased clonogenic survivalrelative to both negative controls (cell line only, and YCFA++DMSO).Soft agar growth of the 451Lu cell line was assessed using the protocolin Materials and Methods, and the results are shown in FIG. 45 .Azacytidine C enhanced soft agar growth inhibition by MRx0029. ERKsignalling in the 451Lu cell line was assessed using the protocol inMaterials and Methods, and the results are shown in FIG. 46 (VEMU, Aza-cand MRx0029 in combination was not assessed). MRx0029 in combinationwith VEMU or Aza-c reduced ERK signalling relative to the negativecontrol (YFCA++DMSO).

These results indicate that MRx0029 alone or in combination withVemurafenib and/or Azacytidine-C has the effects of inducing MAP2 geneexpression, and decreasing clonogenic survival and growth of a melanomacell line carrying a BRAF V600E oncogenic mutation (451Lu). On thisbasis, compositions of the invention are expected to be useful in thetreatment or prevention of cancers, in particular those comprisingoncogenic ERK signalling, especially melanomas such as metastaticmelanomas. In particular, compositions of the invention are expected tobe useful in the treatment or prevention of such cancers comprising anoncogenic mutation in BRAF, in particular at position 600, andespecially the mutation BRAF V600E.

Example 14E—HT29 Colorectal Cancer Cell Line

The effects of the following treatments were assessed on the HT29colorectal cancer cell line (V600E oncogenic mutation in BRAF): (1)MRx0029; (2) Vemurafenib (VEMU) in YFCA+ medium; (3) VEMU and MRx0029;(4) Azacytidine-C (Aza-c) in YCFA+ medium; (5) Aza-c and MRx0029; (6)VEMU, Aza-c and MRx0029.

MAP2 gene expression in the HT29 cell line was assessed using theprotocol in Materials and Methods, and the results are shown in FIG. 47. MRx0029 in combination with VEMU and/or Aza-c increased MAP2 geneexpression relative to both negative controls (cell line only andYCFA+). Clonogenic survival of the HT29 cell line was assessed using theprotocol in Materials and Methods, and the results are shown in FIG. 48. All treatments with MRx0029 (alone or in combination with VEMU and/orAza-c) decreased clonogenic survival relative to both negative controls(cell line only, and YCFA++DMSO). Aza-c improved the effects of MRx0029in inhibiting clonogenic survival. Soft agar growth of the HT29 cellline was assessed using the protocol in Materials and Methods, and theresults are shown in FIGS. 49 a and b . ERK signalling in the HT29 cellline was assessed using the protocol in Materials and Methods, and theresults are shown in FIG. 50 (VEMU, Aza-c and MRx0029 in combination wasnot assessed). MRx0029 alone ERK signalling relative to the negativecontrol (YFCA++DMSO).

These results indicate that MRx0029 alone or in combination withVemurafenib and/or Azacytidine-C has the effects of inducing MAP2 geneexpression, decreasing clonogenic survival and inhibiting ERK signallingin a cell line carrying the V600E oncogenic mutation (HT29). On thisbasis, compositions of the invention are expected to be useful in thetreatment or prevention of cancers, in particular those comprisingoncogenic ERK signalling, especially colorectal cancers such asmetastatic colorectal cancer. In particular, compositions of theinvention are expected to be useful in the treatment or prevention ofsuch cancers comprising an oncogenic mutation in BRAF, in particular atposition 600, and especially the mutation BRAF V600E.

Example 15—GPR109a RNA Expression in Differentiated Caco-2 Cells

GPR109a is a G-protein coupled receptor expressed in the lumen-facingapical membrane of colonic and intestinal epithelial cells. GPR109aexpression silencing is found in colon cancers cell lines, and theinduction of its expression has been reported to induce tumour cellapoptosis in the presence of bacterial fermentation products such asbutyrate [126].

HT29mtx cells seeded on 12 well plates and differentiated for 10 days;then they were serum-starved for 12 hours and subsequently exposed to10% supernatant derived from stationary phase bacteria for 24 h. Cellswere collected, and total RNA was isolated according to the RNeasy minikit protocol (Qiagen). cDNA was made using the high capacity cDNAreverse transcription kit (Applied Biosystems). Gene expression wasmeasured by qPCR. βactin was used as internal control. Fold change wascalculated according to the 2{circumflex over ( )}(−ΔΔct) method [127].The sequences of the forward and reverse primers used are provided asSEQ ID NO: 6 and 7, respectively.

Differentiated Caco-2 form polarised apical/mucosal andbasolateral/serosal membranes that are impermeable and are structurallyand functionally similar to epithelial cells of the small intestine.Treatment of Caco-2 cells with MRx0029 elicited increased expression ofGPR109a (FIG. 52A). Also, Caco-2 treated withphorbol-12-myristate-13-acetate (PMA) supernatant exhibited greaterexpression of GPR109a RNA, than treatment with PMA alone (or PMA inYCFA+ medium)—see FIG. 52B. Therefore, these data suggest thatcompositions of the invention may be useful in the treatment of cancers,especially metastatic cancers, in particular metastatic colorectalcancer or small bowel cancer such as small bowel adenocarcinoma, and inparticular those comprising oncogenic ERK signalling. These data alsosuggest that compositions of the invention may effect such treatmentthrough the mechanism of inducing apoptosis, as a result of GPR109aexpression.

Example 16—Effect of MRx0029 on IL-8 Secretion by the HT29 Cell Line

Differentiated HT29 cells form polarized apical/mucosal andbasolateral/serosal membranes that are impermeable and are structurallyand functionally similar to epithelial cells of the small intestine.

HT29 cells were plated in 12 well plates at a density of 200,000cells/well. Cells were being differentiated for 10 days (media changeevery 2 days). The day of the experiment cells were placed in theanaerobic hood and washed with anaerobic equilibrated HANKs solution.Then 900 ul of growth media (without FBS and antibiotics) was added tothe cells. Bacteria cells were resuspended in of growth media (withoutFBS and antibiotics) and were then added at 10{circumflex over ( )}CFUin total in 100 ul. Cells were co-incubated with bacteria for 2 hr in ananaerobic hood. Afterwards cells were washed in growth media without FBSbut containing antibiotics. Cells were left to rest in 1 ml of THP1condition media for 24 h. After 24 h incubation the supernatant wascollected and spun down at 10,000 g for 3 min and 4° C. Samples werefrozen at −80° C. until further use.

THP1 condition media: THp1 were seeded on T25 flask at density of4×10{circumflex over ( )}6/flask. Cells were treated in RPMI media(contain 2 mM L-glutamine without FBS) with 1 ug/ml LPS or LPS+5 mM ATP(ATP added 3 hours after LPS). Cells were left to rest for 24 hr.Thereafter Condition Media (CM) was collected by spinning down the cellsat 250 g for 5 min and RT. Different CMs were used to treat HT29 Cells.A small aliquot was frozen at 80° C. for ELISA.

Supernatants from the different samples were collected and cytokineanalysis performed according to manufacturer's instruction using a humanIL-8 ELISA kit from Peprotech. GraphPad Prism7 was used to plot andanalyse the data.

MRx0029 increased IL-8 secretion, which is a potent immunostimulatorycytokine (FIGS. 53A and 53B). These data demonstrate theimmunostimulatory activity of MRx0029.

As noted above, secretion of IL-8 increases B cell proliferation. Bcells have been implicated in modulating the immune response to tumours.Indeed, the secretion of anti-tumour antibodies by B cells is a potentmechanisms of tumour control. It is well known that the production oftumour specific antibodies can trigger natural killer cells to bind tothe constant domain of antibodies, resulting in tumor cell lysis throughantibody-dependent cell-mediated cytotoxicity (ADCC). Therefore, thecompositions of the invention may effect the treatment of cancer throughthe appropriate modulation of the B cell response ensuring increasedanti-tumour immune responses.

Based on the fact that the mechanism of pathology of the majority ofcancers involves evasion of surveillance by the host immune system, anymechanism involved in the stimulation of the immune response would havea therapeutically beneficial impact. Therefore, the compositions of theinvention are expected to be useful in the treatment or prevention ofvarious cancers.

Example 17—Metabolite Analysis

Introduction

The gut microbiota, with its immense diversity and metabolic capacity,represents a huge metabolic reservoir for production of a vast varietyof molecules. The inventors sought to determine what short chain fattyacids and medium chain fatty acids are produced and consumed by the M.massiliensis strain NCIMB 42787 and other M. massiliensis strainidentified herein as Ref 1, Ref 2 and Ref 3.

Material and Methods

Bacterial Culture and Cell-Free Supernatant Collection

Pure cultures of bacteria were grown anaerobically in YCFA+ broth untilthey reached their stationary growth phase. Cultures were centrifuged at5,000×g for 5 minutes and the cell-free supernatant (CFS) was filteredusing a 0.2 μM filter (Millipore, UK). 1 mL aliquots of the CFS werestored at −80° C. until use. Sodium butyrate, hexanoic and valeric acidwere obtained from Sigma Aldrich (UK) and suspensions were prepared inYCFA+ broth.

SCFA and MCFA Quantification of Bacterial Supernatants

Short chain fatty acids (SCFAs) and medium chain fatty acids (MCFAs)from bacterial supernatants were analysed and quantified by MS Omics APSas follows. Samples were acidified using hydrochloride acid, anddeuterium labelled internal standards where added. All samples wereanalysed in a randomized order. Analysis was performed using a highpolarity column (Zebron™ ZB-FFAP, GC Cap. Column 30 m×0.25 mm×0.25 μm)installed in a GC (7890B, Agilent) coupled with a quadropole detector(59977B, Agilent). The system was controlled by ChemStation (Agilent).Raw data was converted to netCDF format using Chemstation (Agilent),before the data was imported and processed in Matlab R2014b (Mathworks,Inc.) using the PARADISe software described in [128].

Results

As shown in FIGS. 54-56 , strain 42787 produces valeric acid, butyrateand hexanoic acid and consumes propionate and acetate. The inventorsalso found other strains of the species M. massiliensis that producecomparable levels of valeric acid, hexanoic acid and butyrate and thatconsume similar amounts of acetate and propionate.

Example 18—Suppression of Enolase 2

FIG. 57 demonstrates that MRx0029 has a statistically-significant effectsuppressing neuron specific enolase(NSE)/enolase 2. NSE is thought tosupport increased tumour cell metabolic demands, protect tumour cellsfrom stressful conditions and promote their invasion and migration[129]. It is also implicated in progression of metastatic melanoma[130], survival and progression in small cell lung cancer [131], andprognosis of adenosquamous lung carcinoma [132]. Therefore, thecompositions of the invention are expected to be effective for treatingand preventing cancer, in particular, metastatic melanoma, small celllung cancer and adenosquamous lung carcinoma.

Example 19—Metabolite Analysis

Further to the data provided in Example 17, FIG. 58 demonstrates whatother short chain fatty acids are produced and consumed by the M.massiliensis strain NCIMB 42787 and other strains deposited underaccession numbers NCIMB 43385, NCIMB 43388 and NCIMB 43389.

M. massiliensis strain NCIMB 42787 reduces formic acid while increasinglevels of 2-methyl-propanoic and 3-methyl-butanoic acid (FIG. 58 ).Therefore, strain NCIMB 42787 produces 2-methyl-propanoic and3-methyl-butanoic acid and consumes formic acid. The inventors alsofound that other of the deposited strains produce comparable levels of2-methyl-propanoic and 3-methyl-butanoic acid and consume similaramounts of formic acid.

Example 20—Upregulation of IL-6

Introduction

Bacterial strains were investigated for their ability to trigger anincrease in IL-6 secretion by the astrocytoma cell line U373.

Materials and Methods

Human glioblastoma astrocytoma cell line (U373), were maintained in 25ml MEME 4.5 g/L D-glucose supplemented with 10% heat-inactivated FBS, 4mM L-Glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 5 μg/mlplasmocin, 1% Non-Essential Amino Acids, 1% Sodium Pyruvate (referred toas full growth media).

Cells were plated in 24-well plates at a density of 100,000 cells/wellin 1 ml of full growth media and left to rest at 37° C./5% CO₂ for 72 h.On the day of the treatment, the media was removed from each well, cellswere rinsed with 0.5 ml wash media (serum free MEME), 0.9 ml stimulationmedia (MEME media containing 2% FBS). After 1 h pre-incubation, cellswere removed from CO₂ incubator and treated with 100 μl bacteriasupernatant. YCFA+ media was used as control. Cells were then incubatedfor a further 24 h at 37° C./5% CO₂, after which cell-free supernatantswere collected and spun down at 10,000 g at 4° C. for 3 min. Sampleswere aliquoted in 1.5 ml microtubes and stored in −80° C. for hIL-6ELISA.

Results and Conclusions

FIG. 59 demonstrates that M. massiliensis strain NCIMB 42787 upregulatesIL-6 secretion in U373 cells compared to untreated and YCFA+ controls.Other deposited strains, in particular NCIMB 43389, also increasedsecretion of IL-6. The additional deposited strains are NCIMB 43385,NCIMB 43388, NCIMB 43386 and NCIMB 43387.

Secretion of IL-6 increases B cell proliferation. As outlined above,increased B cell proliferation can act as a potent mechanism forimproving the immune response against a cancer (e.g. via the productionof antibodies and triggering ADCC).

Indeed, the immunostimulatory activity is demonstrated, not only by thedeposited strain, but also by the related deposited strains. Therefore,the compositions of the invention comprising strains of the Megasphaeragenus, or biotypes thereof, are expected to be useful in the treatmentor prevention of various cancers.

Example 21—Suppression of Enolase 2

Materials and Methods

Neuroblastoma cell line SH-SY5Y, were grown in 50% MEM and 50% NutrientMixture F-12 Ham media supplemented with 2 mM L-Glutamine, 10%heat-inactivated FBS, 100 U/ml penicillin and 100 μg/ml streptomycin.SH-SY5Y were plated in 6 well plates at a density of 0.5×10⁶ cells.After 24 h, cells were treated in differentiation medium (growth mediumcontaining 1% FBS) with 10% bacterial supernatants or YCFA+ for 17 h.Cells were collected, and total RNA was isolated according to the RNeasymini kit protocol (Qiagen). cDNA was made using the High Capacity cDNAreverse transcription kit (Applied Biosystems). Gene expression wasmeasured by qPCR. GAPDH was used as internal control. Fold change wascalculated according to the 2^((−ΔΔct)) method. Primer sets used arelisted as SEQ ID NOs: 2, 3, 13 and 14.

Results

FIG. 60 demonstrates M. massiliensis strain NCIMB 42787 has astatistically-significant effect of suppressing neuron specificenolase(NSE)/enolase 2. In addition, the inventors also found depositedreference strains trigger a statistically-significant reduction ofEnolase 2 compared to the YCFA+ culture control. In particular, strainsdeposited under accession numbers NCIMB 43385, NCIMB 43388, NCIMB 43389,NCIMB 43386 and NCIMB 43387 caused a significant suppression of enolase2.

CONCLUSION

Accordingly, in line with the comments in Example 18 above, thecompositions of the invention, in certain embodiments comprising theexemplary reference strains, are expected to be effective for treatingand preventing cancer, in particular, metastatic melanoma, small celllung cancer and adenosquamous lung carcinoma.

Example 22—Upregulation of MAP2

Materials and Methods

Neuroblastoma cell line SH-SY5Y, were grown in 50% MEM and 50% NutrientMixture F-12 Ham media supplemented with 2 mM L-Glutamine, 10%heat-inactivated FBS, 100 U/ml penicillin and 100 μg/ml streptomycin.SH-SY5Y were plated in 6 well plates at a density of 0.5×10⁶ cells.After 24 h, cells were treated in differentiation medium (growth mediumcontaining 1% FBS) with 10% bacterial supernatants or YCFA+ for 17 h.Cells were collected, and total RNA was isolated according to the RNeasymini kit protocol (Qiagen). cDNA was made using the High Capacity cDNAreverse transcription kit (Applied Biosystems). Gene expression wasmeasured by qPCR. GAPDH was used as internal control. Fold change wascalculated according to the 2^((−ΔΔct)) method. Primer sets used arelisted as SEQ ID NOs: 2, 3, 4 and 5.

Results

FIG. 61A displays that M. massiliensis strain NCIMB 42787 and otherdeposited strains trigger a statistically-significant increase of MAP2expression compared to the controls (i.e. negative control and mediacontrol). In particular, strains deposited under accession numbers NCIMB43385, NCIMB 43388, NCIMB 43389, NCIMB 43386 and NCIMB 43387 caused asignificant increase in MAP2 expression. On this basis, compositions ofthe invention are expected to be useful in the treatment or preventionof various cancers, in particular metastatic cancers, in particularmetastatic melanoma.

Example 23—Modulation of Cytokine Secretion in HMC3 Cells Exposed toTNFα Upon Treatment with M. massiliensis Strain NCIMB 42787

Introduction

HMC3 cells were treated with TNFα, and secretion of IL-8 was measuredupon treatment with cell-free supernatants from stationary phase cultureof NCIMB 42787.

Materials and Methods

Human microglia HMC3 cells were grown in glutamine-supplemented EMEMmedia containing 15% heat inactivated FBS and 100 U/ml penicillin and100 μg/ml streptomycin. HMC3 cells were plated in 24 well plates at adensity of 50,000 cells/well. Cells were left in CO₂ incubator to restfor 48 h. The cells were then washed in blank EMEM and pre-treated in 2%FBS growth media with 10 ng/ml TNF-α for 1 h. Thereafter 10% cell-freebacterial supernatants for NCIMB 42787 stationary growth cultures(isolated as described above) were added to TNF-α-treated and untreatedwells and incubated in CO₂ incubator at 37° C. for 24 h. Cell-freesupernatants were collected and centrifugated at 10,000×g for 3 min and4° C. Samples were aliquoted in 1.5 ml microtubes and stored in −80° C.for hIL-8 ELISA.

Secretion of IL-8 was analysed using hIL-8 Standard ELISA Kits,according to the manufacturer's protocol in the cell-free supernatantsfrom HMC3 cells treated as described above. Samples were measured at 405nm with correction wavelength set at 655 nm on a microplate reader(iMark, Bio-Rad). Raw data were plotted and analysed using GraphPadPrism 7 software.

Statistical Analysis

Normally distributed data are presented as mean±SEM; One-way Anova(Sidak's multiple comparison test) was used to analyse the datapresented in this paper. A p value <0.05 was deemed significant in allcases.

Results

NCIMB 42787 induces IL-8 secretion in the absence of stimulation (FIG.61B). As outlined above, IL-8 is involved in the activation of theimmune system, in particular by stimulation of B cell proliferation.

Example 24—NF-κB Promoter Activation in HEK-TLR4 Cells by M.massiliensis NCIMB 42787

Introduction

To verify whether treatment with NCIMB 42787 would induce NF-κB-Ap1promoter activity induced by engagement of TLR4, HEK-TLR4 cells weretreated with cell-free bacterial supernatants for NCIMB 42787 alone orin combination with LPS.

Materials and Methods

HEK293-Blue reporter cells stably expressing human TLR4 (HEK-TLR4), werecultured according to the manufacturer's instructions. Briefly, HEK-TLR4cells were maintained in DMEM 4.5 g/L D-glucose supplemented with 10%(v/v) heat-inactivated FBS, 4 mM L-Glutamine, 100 U/ml penicillin, 100μg/ml streptomycin, 100 μg/ml normocin, 1×HEK-Blue selection media.

Briefly, cells were washed with PBS, dissociated in PBS and collected ingrowth media. Cells were plated in 96-well plates at a density of 25,000cells/well. To evaluate the effect of bacteria strains on LPS inducingNF-κB promoter activation, cells were treated with 10 ng/ml LPS inpresence or absence of 10% supernatants (isolated as described above)and incubated in a CO₂ incubator. Treatments proceeded for 22 h at 37°C. and 5% CO, after which the detection of Secreted Embryonic AlkalinePhosphatase (SEAP) activity from cell culture supernatant was performedusing QUANTI-blue solution according to manufacturer's instructions.Briefly, 20 μl of cell-free supernatant was collected and analysed forthe presence of SEAP by mixing with 200 μl of sterile-filteredQUANTI-Blue detection media. After 2 h incubation at 37° C., opticaldensity was measured at 655 nm on a microplate reader (iMark microplate,Bio-Rad).

Statistical Analysis

Normally distributed data are presented as mean±SEM; One-way Anova(Sidak's multiple comparison test) was used to analyse the datapresented in this paper. A p value <0.05 was deemed significant in allcases.

Results

NCIMB 42787 induced NF-κB-Ap1 promoter activation on its own (FIG. 61C).

NF-κB is involved in the activation of the immune response in particularby stimulating the expression of mediators of inflammation and cytokinesinvolved in the immune response, for example IL-6. As outlined above, anincrease in the expression of IL-6 helps to stimulate the immune systemand thus the activation of the NF-κB pathway has immunostimulatoryactivity. Accordingly, in certain embodiments, the compositions of theinvention activate NF-κB signalling and thus stimulate the immunesystem.

Example 25—M. massiliensis Strains Produce Butyric, Valeric and HexanoicAcid

Materials and Methods

SCFA extraction from YCFA+, YCFA+ spiked with a standard mix of SCFAs(40 mM acetic acid and 20 mM formic acid, propionic acid, butyric acid,valeric acid and hexanoic acid) was conducted according to the method ofDe Baere et al.¹³³

HPLC Analysis of SCFAs

HPLC detection and quantification of SCFAs was conducted according tothe method of De Baere et al.¹³³ with slight modifications. Briefly,HPLC analysis was performed using a Waters e2695 HPLC system equippedwith a Waters Photodiode Array (PDA) detector 2998 (Waters Limited,Elstree, UK). HPLC analysis of SCFAs standards, SCFAs extracted fromMRx0005 and MRx0029 BCFS and MRx0005 and MRx0029 hexane, diethyl ether,ethyl acetate, acetonitrile and methanol extracts were performed usingan Xselect® HSS T3 3.5 μm 4.6×150 mm LC column (Waters Limited, Elstree,UK). The LC analysis was performed using the photodiode array detector(PDA) set to analyse wavelengths of 200-800 nm. SCFA detection andquantification was performed at 210 nm. The mobile phase consisted in 25mM sodium phosphate buffer in HPLC water (pH adjusted to 3.0 usingphosphoric acid (A) and acetonitrile (B). The LC method for SCFAdetection and quantification was run using the solvent system with thefollowing gradient: t0′ A=95%, B=5%; t10′ A=95%, B=5%; t30′ A=30%,B=70%; t31′ A=0%, B=100%; t36′ A=0%, B=100%; t38′ A=5%, B=95%; t60′A=5%, B=95%; flow=1 ml/min.

A seven-point calibration curve was prepared for each SCFA by injecting20 μl of a two-fold serial dilution of a SCFA (40 mM acetic acid and 20mM formic acid, propionic acid, butyric acid, valeric acid and hexanoicacid). Quantification-extraction efficiency was calculated using theformula below:

[SCFA in YCFA+spiked and extracted]/[SCFA in YCFA+spiked not extracted]

Extraction efficiency was used to determine the concentrations ofindividual SCFAs in each sample. The production of specific SCFAs wascalculated by subtracting the amount of corresponding SCFA present inthe unspiked media control.

Targeted Metabolomics: Bacterial Metabolites and Fatty Acid Analysis

Sample analysis was carried out by MS-Omics (Copenhagen, Denmark). Amixed pooled sample (QC sample) was created by taking an aliquot fromeach sample. This sample was analysed with regular intervals throughoutthe sequence. Matrix effects were tested for quantified compounds byspiking the QC sample in a minimum of two levels.

For GC-metabolite analysis, samples were derivatized with methylchloroformate using a slightly modified version of the protocoldescribed by Smart et al.¹³⁴. All samples were analysed in a randomizedorder. Analysis was performed using GC (7890B, Agilent) coupled with aquadrupole detector (59977B, Agilent). Raw data was converted to netCDFformat using Chemstation (Agilent), before the data was imported andprocessed in Matlab R2014b (Mathworks, Inc.) using the PARADISe softwaredescribed by Johnsen et al.¹³⁵.

For SCFA analysis, samples were acidified using hydrochloric acid, anddeuterium-labelled internal standards were added. Analysis was performedusing a high-polarity column (Zebron™ ZB-FFAP, GC Cap. Column 30 m×0.25mm×0.25 μm) installed in a GC (7890B, Agilent) coupled with a quadrupoledetector (59977B, Agilent). Raw data was converted to netCDF formatusing Chemstation (Agilent), before the data was imported and processedin Matlab R2014b (Mathworks, Inc.) using the PARADISe software describedby Johnsen et al.¹³⁵.

Results

Fatty acid analysis, using targeted metabolomics, demonstrated thatNCIMB 42787 produces butanoic (butyric), pentanoic (valeric) andhexanoic (caproic) acid, both in the linear and branched forms (C4-C6)(FIG. 62A). Moreover, the ratio of 4-hydroxy-phenylacetic acid:media wasincreased in NCIMB 42787 cell-free supernatant. HPLC analysis ofcell-free supernatants was used to monitor the production of formic,acetic, propionic, butyric, valeric, and hexanoic acid (based onretention time and absorbance spectrum of relevant SCFAs) by NCIMB42787. Representative chromatograms for SCFA standards overlaid to NCIMB42787 cell-free supernatants extracted for SCFAs are reported in FIG.62C. HPLC analysis confirmed the production of butyric, valeric andhexanoic acid by NCIMB 42787.

Example 26—M. Massiliensis Methanolic Fractions Containing Butyrate andValerate Show Immunostimulatory Activity in U373 Cells

To investigate the role of SCFAs in reducing secretion of IL-8, U373cells were treated with increasing concentrations of sodium butyrate(SB), sodium valerate (SV) and hexanoic acid (HA).

Methods

U373 cells were prepared as described above. Cells were pre-treated for1 h with 1 μg/ml LPS indicated above and incubated at 37° C. and 5% CO₂.After 1 h pre-incubation, cells were removed from CO₂ incubator andtreated with increasing concentration of fresh prepared Sodium Butyrate(SB), Sodium Valerate (SV) and Hexanoic Acid (HA).

Statistical Analysis

Normally distributed data are presented as mean±SEM; One-way Anova(Sidak's multiple comparison test) was used to analyse the datapresented in this paper. A p value <0.05 was deemed significant in allcases.

Results and Conclusions

The concentrations tested covered the range of concentrations measuredin the cell-free supernatants for the different fatty acids and tookinto account the fact that only 10% of the above-mentioned supernatantswas used in the cell-based assays. Both SB and SV increased LPS-inducedsecretion of IL-8 in the same cells (FIG. 63 ), suggesting that thepresence of both SCFAs likely contributed to IL-8 induction when NCIMB42787 is added to the culture. HA did not inhibit IL-8 secretion afterchallenge with LPS. None of the SFCAs tested induced per se secretion ofIL-8 above the basal level (untreated cell control). The reconstitutedmixture of the three SCFAs reproduced the biological activity of NCIMB42787 cell-free supernatant, both in the presence and absence of LPS.

Accordingly, in certain embodiments butyric and/or valeric acid areinvolved in the generation of IL-8, and therefore are important forstimulating the immune system, for example via B cell proliferation.Therefore, in certain embodiments, the bacterial strains of theinvention stimulate the immune system via the production of butyricand/or valeric acid.

Example 27—SCFAs Generated by NCIMB 42787 are at Least PartiallyResponsible for Immunostimulatory Activity

Introduction

In order to further confirm whether the activity of NCIMB 42787 was dueat least in part to SCFAs, cell-free bacterial supernatant wasfractionated with different solvents of increasing polarity. HPLCanalysis of the de-proteinased crude extracts (hexane, F5; diethylether, F4; ethyl acetate, F3; acetonitrile, F4; methanol, F1) of thisstrain supernatants was conducted to analyse the biochemical complexityof the stationary phase cell-free supernatants of NCIMB 42787, as wellas to sub-fractionate compounds based on polarity and solubility.

Methods

Sequential Solvent Extractions—Preparation of Crude Extracts

Three biological replicates of NCIMB 42787 strain BCFSs and YCFA+(mediacontrol) were extracted sequentially with HPLC-grade hexane (HEX),diethyl ether (DE), ethyl acetate (EtOAc), acetonitrile (ACN) andmethanol (MeOH). Briefly, 20 ml of BCFS were placed in glass vials andextracted at room temperature (RT) in 20 ml of HEX on a rotary shaker(70 rpm) for 30 min. A total of three extractions were performed on eachBCFS and YCFA+ media control. The remaining aqueous layers were thenextracted at RT in 20 ml of DE, EtOAc on a MX-RD-Pro rotary shaker (70rpm) for 30 min a total of three times. The combined extracts of eachsample were dried under reduced pressure in an R-300 rotary evaporatorequipped with a V-300 vacuum pump (Büchi, Flawil, Switzerland) at atemperature not exceeding 30° C. The resulting extracts werere-solubilised in 2 ml of corresponding solvent and aliquoted in four1.5 ml Eppendorf tubes (500 μl each corresponding to 5 ml of originalsample). The remaining aqueous layers were then extracted at RT in 20 mlof DE, EtOAc on a MX-RD-Pro rotary shaker (70 rpm) for 30 min a total ofthree times. The combined extracts of each sample were dried underreduced pressure in a R-300 rotary evaporator equipped with a V-300vacuum pump (Büchi, Flawil, Switzerland) at a temperature not exceeding30° C. The resulting extracts were re-solubilised in 2 ml ofcorresponding solvent and aliquoted in four 1.5 ml Eppendorf tubes (500μl each corresponding to 5 ml of original sample).

The remaining aqueous layers were evaporated to dryness using an R-300rotary evaporator. The resulting dry extracts were extracted for 30 minin 20 ml of ACN a total of three times. The ACN extracts were combined,evaporated to dryness using a rotary evaporator, resolubilised in 2 mlof ACN and aliquoted in four 1.5 ml Eppendorf tubes (500 μl each). Theremaining dry extracts (ACN insoluble portion of the extracts) were thenextracted for 30 min in 20 ml of MeOH a total of three times. The MeOHextracts were combined, evaporated to dryness using an R-300 RotaryEvaporator, resolubilised in 2 ml of MeOH and aliquoted in four 1.5 mlEppendorf tubes (500 μl each).

Aliquots of the crude extracts were kept overnight at −20° C. inducingthe precipitation of proteinaceous components. Following overnightprecipitation, each aliquot was centrifuged at 10,000×g for 6 min andtransferred to a new 2 ml tube. Overnight precipitation was repeatedthree times after which extracts were dried in a RVC 2-18 CDPlusspeedvac (Christ, Osterode am Harz, Germany) and weighed. All driedaliquots of each extract were stored at −80° C. until further use.

Treatment

U373 cells were prepared as described above. Cells were pre-treated for1 h with 1 μg/ml LPS as indicated above. Afterwards, cells were removedfrom CO₂ incubator and treated with 100 μl of the different fractions.Fractions from media were used as controls. Cell-free supernatants werecollected 24 h after treatment and analysed by ELISA for IL-8 secretion(as outlined above).

Statistical Analysis

Normally distributed data are presented as mean±SEM; One-way Anova(Sidak's multiple comparison test) was used to analyse the datapresented in this paper. A p value <0.05 was deemed significant in allcases.

Results

HPLC analysis confirmed the selective extraction and crude fractionationof compounds present in the de-proteinased supernatants. Theunfractionated NCIMB 42787 induced IL-8 secretion in U373 cells both inthe presence and absence of LPS, and the same activity was produced bythe methanolic fraction F1, thus reiterating the important role ofbutyric and valeric acid in IL-8 production by these cell-types (FIGS.64A and 64B).

Therefore, as outlined above, in certain embodiments, the production ofbutyric and/or valeric acid stimulated the immune system. Accordingly,in certain embodiments, the bacterial strains of the invention stimulatethe immune system via the production of butyric and/or valeric acid.

Example 28—Megasphaera Reference Strain NCIMB 43387 SignificantlyReduces Colonic IDO-1 mRNA Expression in BALB/c Mice

FIG. 65 demonstrates that NCIMB 43387 causes a significant reduction(quantified by qPCR normalised to β-actin) in IDO-1 mRNA expression inthe colon of BALB/c mice compared with the vehicle control.

IDO-1 has been implicated in promoting immunosuppression in response toinflammation or infection. Accordingly, driving a decrease in IDO-1expression is associated with immunostimulation. Furthermore, reductionsin IDO-1 reduce the proliferatory and migratory capacity of cancercells, and improves immune surveillance against tumours. Therefore, incertain embodiments, the bacterial strains of the invention serve toreduce IDO-1 expression. In certain embodiments, immunostimulation isassociated with reduction of IDO-1 expression. In addition, in certainembodiments, the compositions of the present invention preventmetastatic cancer growth. In certain embodiments, the compositions ofthe present invention are effective for treating and preventing cancer,in particular, metastatic melanoma, small cell lung cancer andadenosquamous lung carcinoma in light of their activity againstmetastasis.

Example 29—Megasphaera Strains Deposited Under Accession Numbers NCIMB43385 and NCIMB 43387 Reduce Colonic Tph-1 mRNA Expression in BALB/cMice

BALB/c mice were administered live biotherapeutic and tissues wereisolated for analysis of gene expression using qPCR.

FIG. 66 demonstrates the ability of the compositions of the invention toreduce expression of Tph-1 mRNA (using quantification by qPCR normalisedto β-actin) compared to the vehicle control.

Decreases in Tph-1 are known to be associated with increased cancerresistance and reduced cancer cell growth. In addition, a decrease inTph-1 activity stimulates the immune system by providing sufficientlevels of the essential amino acid tryptophan for mast cells to driveanti-tumour immunity. Accordingly, in certain embodiments, thecompositions of the invention decrease the levels of Tph-1 expression.In certain embodiments, the compositions of the invention triggerimmunostimulation and treat and/or prevent the diseases disclosed hereinby reducing the levels of Tph-1.

Example 30—Megasphaera Strain Deposited Under Accession Number NCIMB43385 Increases IFNγ and IL-6 Production Upon ConA Stimulation ofSplenocytes from BALB/c Mice

Live biotherapeutic strains were screened ex vivo for efficacy of immunemarker production in splenocytes isolated from BALB/c mice andstimulated with ConA.

FIGS. 67A and 67B displays the ability of compositions of the inventionto significantly increase production of the pro-inflammatory cytokinesIFNγ and IL-6. As outlined above, both of these cytokines are involvedin the stimulation of the immune response. Furthermore, IFNγ hassignificant tumoricidal activity.

Accordingly, as outlined above, in certain embodiments, the compositionsof the invention increase the production of IFNγ and/or IL-6 andtherefore drive stimulation of the immune response. Accordingly, incertain embodiments, the therapeutic benefit of the compositions of thepresent invention is linked to an increase in IFNγ and/or IL-6production.

Example 30—Megasphaera Reference Strain Deposited Under Accession NumberNCIMB 43385 Significantly Increases IL-6 and CD11b Expression

BALB/c mice were administered live biotherapeutic and tissues wereisolated for analysis of gene expression using qPCR.

FIG. 68 demonstrates the ability of NCIMB 43385 to significantlyincrease IL-6 and CD11b expression in the hippocampus of BALB/c micecompared to vehicle control.

Accordingly, as outlined above, the compositions of the presentinvention, in certain embodiments increase the expression ofpro-inflammatory cytokines involved in the stimulation of the immuneresponse, in particular IL-6 and CD11b. In certain embodiments, thecompositions of the invention are therapeutically effective in light ofthe increase in IL-6 and CD11b expression.

Example 31—NCIMB 42787 Increases TLR4 Expression

BALB/c mice were administered live biotherapeutic and tissues wereisolated for analysis of gene expression using qPCR.

FIG. 69 demonstrates the ability of NCIMB 42787 to significantlyincrease TLR4 expression in the amygdala of BALB/c mice compared tovehicle control.

TLR4 is associated with activating the immune response. Accordingly,increasing TLR4 expression will improve immunostimulation. In certainembodiments, the compositions of the invention increase expression ofTLR4. In certain embodiments, the increase in TLR4 expression increasesthe immune response. In certain embodiments, the compositions of thepresent invention increase the immune response and are therapeuticallyeffect in treating the diseases disclosed herein via the increase inTLR4 expression.

Sequences

(consensus 16S rRNA sequence forMegasphaera massiliensis strain MRx0029) SEQ ID NO: 1TGAGAAGCTTGCTTCTTATCGATTCTAGTGGCAAACGGGTGAGTAACGCGTAAGCAACCTGCCCTTCAGATGGGGACAACAGCTGGAAACGGCTGCTAATACCGAATACGTTCTTTCCGCCGCATGACGGGAAGAAGAAAGGGAGGCCTTCGGGCTTTCGCTGGAGGAGGGGCTTGCGTCTGATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGTCTGAGAGGATGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAACGATGACGGCCTTCGGGTTGTAAAGTTCTGTTATATGGGACGAACAGGACATCGGTTAATACCCGGTGTCTTTGACGGTACCGTAAGAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCGCGCAGGCGGCATCGCAAGTCGGTCTTAAAAGTGCGGGGCTTAACCCCGTGAGGGGACCGAAACTGTGAAGCTCGAGTGTCGGAGAGGAAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAAGCGGCTTTCTGGACGACAACTGACGCTGAGGCGCGAAAGCCAGGGGAGCAAACGGGATTAGATACCCCGGTAGTCCTGGCCGTAAACGATGGATACTAGGTGTAGGAGGTATCGACTCCTTCTGTGCCGGAGTTAACGCAATAAGTATCCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGACGCAACGCGAAGAACCTTACCAAGCCTTGACATTGATTGCTACGGAAAGAGATTTCCGGTTCTTCTTCGGAAGACAAGAAAACAGGTGGTGCACGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTCTGTTGCCAGCACCTCGGGTGGGGACTCAGAAGAGACTGCCGCAGACAATGCGGAGGAAGGCGGGGATGACGTCAAGTCATCATGCCCCTTATGGCTTGGGCTACACACGTACTACAATGGCTCTTAATAGAGGGAAGCGAAGGAGCGATCCGGAGCAAACCCCAAAAACAGAGTCCCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCAGGAATCGCTAGTAATCGCAGGTCAGCATACTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAAAGTCATTCACACCCGAAGCCGGTGAGGCAACCGCAAG

Name Forward sequence Reverse sequence GAPDH GGTATCGTGGAAGGACTCATGATGCCAGTGAGCTTCCCGTTC (SEQ ID NO: 2) (SEQ ID NO: 3) MAP2CTCAGCACCGCTAACAGAGG CATTGGCGCTTCTCTCCTC (SEQ ID NO: 4) (SEQ ID NO: 5)GPR109a ATGTTGGCTATGAACCGCCAG GCTGCTGTCCGATTGGAGA (SEQ ID NO: 6)(SEQ ID NO: 7)

(consensus 16S rRNA sequence for the Megasphaera strain deposited underaccession number NCIMB 43385) SEQ ID NO: 8GGCTGGTTCCTTGCGGTTGCCTCACCGGCTTCGGGTGTGAATGACTTTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGTATGCTGACCTGCGATTACTAGCGATTCCTGCTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGGGACTCTGTTTTTGGGGTTTGCTCCGGATCGCTCCTTCGCTTCCCTCTATTAAGAGCCATTGTAGTACGTGTGTAGCCCAAGCCATAAGGGGCATGATGACTTGACGTCATCCCCGCCTTCCTCCGCATTGTCTGCGGCAGTCTCTTCTGAGTCCCCACCCTTAGTGCTGGCAACAGAAGATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCGTGCACCACCTGTTTTCTTGTCTTCCGAAGAAGAACCGGAAATCTCTTTCCGTAGCAATCAATGTCAAGGCTTGGTAAGGTTCTTCGCGTTGCGTCGAATTAAACCACATACTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGGATACTTATTGCGTTAACTCCGGCACAGAAGGAGTCGATACCTCCTACACCTAGTATCCATCGTTTACGGCCAGGACTACCGGGGTATCTAATCCCGTTTGCTCCCCTGGCTTTCGCGCCTCAGCGTCAGTTGTCGTCCAGAAAGCCGCTTTCGCCACTGGTGTTCCTCCTAATATCTACGCATTTCACCGCTACACTAGGAATTCCGCTTTCCTCTCCGACACTCGAGCTTCACAGTTTCGGTCCCCTCACGGGGTTAAGCCCCGCACTTTTAAGACCGACTTGCGATGCCGCCTGCGCGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTCTTACGGTACCGTCAGGGATAACGGGTATTGACCGCTATCCTGTTCGTCCCATATAACAGAACTTTACAACCCGAAGGCCGTCATCGTTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAATGTGGCCGTTCATCCTCTCAGACCGGCTACTGATCGTCGCCTTGGTGGGCCGTTACCCCTCCAACTAGCTAATCAGACGCAAGCCCCTCCTCCAGCGAAAGCCCGAAGGCCTCCCTTTCTTCATCCCGTCATGCGGCGGAAAGAACGTATTCGGTATTAGCAGCCGTTTCCAGCTGTTGTCCCCATCTGAAGGGCAGGTTGCTTACGCGTTACTCACCCGTTTGCCACTCGAATTGATAAGAAGCAAGCTTCTCATC(consensus 16S rRNA sequence for the Megasphaera massilliensis straindeposited under accession number NCIMB 43388) SEQ ID NO: 9GGCTGGTTCCTTGCGGTTGCCTCACCGGCTTCGGGTGTGAATGACTTTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGTATGCTGACCTGCGATTACTAGCGATTCCTGCTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGGGACTCTGTTTTTGGGGTTTGCTCCGGATCGCTCCTTCGCTTCCCTCTATTAAGAGCCATTGTAGTACGTGTGTAGCCCAAGCCATAAGGGGCATGATGACTTGACGTCATCCCCGCCTTCCTCCGCATTGTCTGCGGCAGTCTCTTCTGAGTCCCCACCCGAGGTGCTGGCAACAGAAGATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCGTGCACCACCTGTTTTCTTGTCTTCCGAAGAAGAACCGGAAATCTCTTTCCGTAGCAATCAATGTCAAGGCTTGGTAAGGTTCTTCGCGTTGCGTCGAATTAAACCACATACTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGGATACTTATTGCGTTAACTCCGGCACAGAAGGAGTCGATACCTCCTACACCTAGTATCCATCGTTTACGGCCAGGACTACCGGGGTATCTAATCCCGTTTGCTCCCCTGGCTTTCGCGCCTCAGCGTCAGTTGTCGTCCAGAAAGCCGCTTTCGCCACTGGTGTTCCTCCTAATATCTACGCATTTCACCGCTACACTAGGAATTCCGCTTTCCTCTCCGACACTCGAGCTTCACAGTTTCGGTCCCCTCACGGGGTTAAGCCCCGCACTTTTAAGACCGACTTGCGATGCCGCCTGCGCGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTCTTACGGTACCGTCAAAGACACCGGGTATTAACCGATGTCCTGTTCGTCCCATATAACAGAACTTTACAACCCGAAGGCCGTCATCGTTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAATGTGGCCGTTCATCCTCTCAGACCGGCTACTGATCGTCGCCTTGGTGGGCCGTTACCCCTCCAACTAGCTAATCAGACGCAAGCCCCTCCTCCAGCGAAAGCCCGAAGGCCTCCCTTTCTTCTTCCCGTCATGCGGCGGAAAGAACGTATTCGGTATTAGCAGCCGTTTCCAGCTGTTGTCCCCATCTGAAGGGCAGGTTGCTTACGCGTTACTCACCCGTTTGCCACTAGAATCGATAAGAAGCAAGCTTCTCATGTCTTCT(consensus 16S rRNA sequence for the Megasphaera massilliensisstrain deposited under accession number NCIMB 43389) SEQ ID NO: 10CGACGGCTGGTTCCTTGCGGTTGCCTCACCGGCTTCGGGTGTGAATGACTTTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGTATGCTGACCTGCGATTACTAGCGATTCCTGCTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGGGACTCTGTTTTTGGGGTTTGCTCCGGATCGCTCCTTCGCTTCCCTCTATTAAGAGCCATTGTAGTACGTGTGTAGCCCAAGCCATAAGGGGCATGATGACTTGACGTCATCCCCGCCTTCCTCCGCATTGTCTGCGGCAGTCTCTTCTGAGTCCCCACCCGAGGTGCTGGCAACAGAAGATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCGTGCACCACCTGTTTTCTTGTCTTCCGAAGAAGAACCGGAAATCTCTTTCCGTAGCAATCAATGTCAAGGCTTGGTAAGGTTCTTCGCGTTGCGTCGAATTAAACCACATACTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGGATACTTATTGCGTTAACTCCGGCACAGAAGGAGTCGATACCTCCTACACCTAGTATCCATCGTTTACGGCCAGGACTACCGGGGTATCTAATCCCGTTTGCTCCCCTGGCTTTCGCGCCTCAGCGTCAGTTGTCGTCCAGAAAGCCGCTTTCGCCACTGGTGTTCCTCCTAATATCTACGCATTTCACCGCTACACTAGGAATTCCGCTTTCCTCTCCGACACTCGAGCTTCACAGTTTCGGTCCCCTCACGGGGTTAAGCCCCGCACTTTTAAGACCGACTTGCGATGCCGCCTGCGCGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTCTTACGGTACCGTCAAAGACACCGGGTATTAACCGATGCCCTGTTCGTCCCATATAACAGAACTTTACAACCCGAAGGCCGTCATCGTTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAATGTGGCCGTTCATCCTCTCAGACCGGCTACTGATCGTCGCCTTGGTGGGCCGTTACCCCTCCAACCAGCTAATCAGACGCAAGCCCCTCCTCCAGCGAAAGCCCGAAGGCCTCCCTTTCTTCTTCCCGTCATGCGGCGGAAAGAACGTATTCGGTATTAGCAGCCGTTTCCAGCTGTTGTCCCCATCTGAAGGGCAGGTTGCTTACGCGTTACTCACCCGTTTGCCACTAGAATCGATAAGAAGCAAGCTTCTCATGTCTTCTCGTTCGACTTGCAT(consensus 16S rRNA sequence for the Megasphaera strain deposited underaccession number NCIMB 43386) SEQ ID NO: 11CGACGGCTGGTTCCTTGCGGTTGCCTCACCGGCTTCGGGTGTGAATGACTTTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGTATGCTGACCTGCGATTACTAGCGATTCCTGCTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGGGACTCTGTTTTTGGGGTTTGCTCCGGATCGCTCCTTCGCTTCCCTCTATTAAGAGCCATTGTAGTACGTGTGTAGCCCAAGCCATAAGGGGCATGATGACTTGACGTCATCCCCGCCTTCCTCCGCATTGTCTGCGGCAGTCTCTTCTGAGTCCCCACCCTTAGTGCTGGCAACAGAAGATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCGTGCACCACCTGTTTTCTTGTCTTCCGAAGAAGAACCGGAAATCTCTTTCCGTAGCAATCAATGTCAAGGCTTGGTAAGGTTCTTCGCGTTGCGTCGAATTAAACCACATACTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGGATACTTATTGCGTTAACTCCGGCACAGAAGGAGTCGATACCTCCTACACCTAGTATCCATCGTTTACGGCCAGGACTACCGGGGTATCTAATCCCGTTTGCTCCCCTGGCTTTCGCGCCTCAGCGTCAGTTGTCGTCCAGAAAGCCGCTTTCGCCACTGGTGTTCCTCCTAATATCTACGCATTTCACCGCTACACTAGGAATTCCGCTTTCCTCTCCGACACTCGAGCTTCACAGTTTCGGTCCCCTCACGGGGTTAAGCCCCGCACTTTTAAGACCGACTTGCGATGCCGCCTGCGCGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTCTTACGGTACCGTCAGGGATAACGGGTATTGACCGCTATCCTGTTCGTCCCATATAACAGAACTTTACAACCCGAAGGCCGTCATCGTTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAATGTGGCCGTTCATCCTCTCAGACCGGCTACTGATCGTCGCCTTGGTGGGCCGTTACCCCTCCAACTAGCTAATCAGACGCAAGCCCCTCCTCCAGCGAAAGCCCGAAGGCCTCCCTTTCTTCATCCCGTCATGCGGCGGAAAGAACGTATTCGGTATTAGCAGCCGTTTCCAGCTGTTGTCCCCATCTGAAGGGCAGGTTGCTTACGCGTTACTCACCCGTTTGCCACTCGAATTGATAAGAAGCAAGCTTCTCATCTCTTCTCGTTCGACTGCA(consensus 16S rRNA sequence for the Megasphaera strain deposited underaccession number NCIMB 43387) SEQ ID NO: 12TCGAACGGCTGGTTCCTTGCGGTTGCCTCACCGGCTTCGGGTGTGAATGACTTTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGTATGCTGACCTGCGATTACTAGCGATTCCTGCTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGGGACTCTGTTTTTGGGGTTTGCTCCGGATCGCTCCTTCGCTTCCCTCTATTAAGAGCCATTGTAGTACGTGTGTAGCCCAAGCCATAAGGGGCATGATGACTTGACGTCATCCCCGCCTTCCTCCGCATTGTCTGCGGCAGTCTCTTCTGAGTCCCCACCCTTAGTGCTGGCAACAGAAGATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCGTGCACCACCTGTTTTCTTGTCTTCCGAAGAAGAACCGGAAATCTCTTTCCGTAGCAATCAATGTCAAGGCTTGGTAAGGTTCTTCGCGTTGCGTCGAATTAAACCACATACTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGGATACTTATTGCGTTAACTCCGGCACAGAAGGAGTCGATACCTCCTACACCTAGTATCCATCGTTTACGGCCAGGACTACCGGGGTATCTAATCCCGTTTGCTCCCCTGGCTTTCGCGCCTCAGCGTCAGTTGTCGTCCAGAAAGCCGCTTTCGCCACTGGTGTTCCTCCTAATATCTACGCATTTCACCGCTACACTAGGAATTCCGCTTTCCTCTCCGACACTCGAGCTTCACAGTTTCGGTCCCCTCACGGGGTTAAGCCCCGCACTTTTAAGACCGACTTGCGATGCCGCCTGCGCGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTCTTACGGTACCGTCAGGGATAACGGGTATTGACCGCTATCCTGTTCGTCCCATATAACAGAACTTTACAACCCGAAGGCCGTCATCGTTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAATGTGGCCGTTCATCCTCTCAGACCGGCTACTGATCGTCGCCTTGGTGGGCCGTTACCCCTCCAACTAGCTAATCAGACGCAAGCCCCTCCTCCAGCGAAAGCCCGAAGGCCTCCCTTTCTTCATCCCGTCATGCGGCGGAAAGAACGTATTCGGTATTAGCAGCCGTTTCCAGCTGTTGTCCCCATCTGAAGGGCAGGTTGCTTACGCGTTACTCACCCGTTTGCCACTCGAATTGATAAGAAGCAAGCTTCTCATCTCTTCTCGTTCGACTTGCA

Primers Used for qPCR of Enolase

Name Forward sequence Reverse sequence NSE CCCTGTATCGTAAGAACGGTGCCACCATTGATCACGTTGA (SEQ ID NO: 13) (SEQ ID NO: 14)

(biotype identifying repetitive sequence) SEQ ID NO: 15 GTGGTGGTGG TGGT

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1.-28. (canceled)
 29. A method of stimulating an immune system in a subject in need thereof, the method comprising administering to said subject a pharmaceutical composition comprising a therapeutically effective amount of a bacteria strain of the genus Megasphaera, wherein said bacteria strain comprises a 16S rRNA gene sequence that has at least 95% sequence identity to the polynucleotide sequence of SEQ ID NO: 1, wherein said subject has (i) reduced immune response or (ii) a disease or condition associated with reduced immune response, wherein said administering is effective to increase immune response in said subject compared to prior to said administering, thereby stimulating said immune system.
 30. The method of claim 29, wherein said administering selectively decreases the number and/or percentage of regulatory T (Treg) cells in a cell population in said subject.
 31. The method of claim 30, wherein said cell population comprises peripheral blood mononuclear cells (PBMCs).
 32. The method of claim 29, wherein said administering enhances secretion of one or more cytokines.
 33. The method of claim 32, wherein said one or more cytokines comprise IL-1β, TNF-α, MIP-3α, IL-23, IL-8, or IL-6.
 34. The method of claim 29, wherein said therapeutically effective amount of said bacteria strain comprises at least about 1×10³ colony forming units per gram (CFU/g) of said bacteria strain with respect to the total weight of said pharmaceutical composition.
 35. The method of claim 29, wherein said therapeutically effective amount of said bacteria strain comprises from about 1×10³ to about 1×10¹¹ colony forming units per gram (CFU/g) of said bacteria strain with respect to the total weight of said pharmaceutical composition.
 36. The method of claim 29, wherein said pharmaceutical composition is formulated for oral, rectal, nasal, buccal, sublingual, or subcutaneous administration.
 37. The method of claim 29, wherein said bacterial strain is lyophilized.
 38. The method of claim 29, wherein said pharmaceutical composition is formulated for delivery to an intestine of said subject.
 39. The method of claim 29, wherein said bacteria strain is capable of at least partially colonizing an intestine of said subject.
 40. The method of claim 29, wherein said pharmaceutical composition is encapsulated.
 41. The method of claim 29, wherein said pharmaceutical composition is formulated in a form of tablet, capsule, or powder.
 42. The method of claim 29, wherein said at least 95% sequence identity is determined by a Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of
 2. 43. The method of claim 29, wherein said bacteria strain comprises a 16S rRNA gene sequence that has at least 98% sequence identity to the polynucleotide sequence of SEQ ID NO:
 1. 44. The method of claim 29, wherein said bacteria strain comprises a 16S rRNA gene sequence that has at least 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 1
 45. The method of claim 29, wherein said bacteria strain comprises a 16S rRNA gene sequence that is the polynucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:
 12. 46. The method of claim 29, wherein said bacterial strain is the strain deposited under accession number NCIMB 42787, NCIMB 43385, NCIMB 43386, NCIMB 43387, NCIMB 43388, or NCIMB
 43389. 47. The method of claim 29, wherein said bacterial strain is of the species Megasphaera massiliensis.
 48. The method of claim 29, wherein said pharmaceutical composition further comprises a pharmaceutically acceptable excipient, carrier, or diluent. 